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Fungal Cellulases
Christina M. Payne,†,# Brandon C. Knott,‡,# Heather B. Mayes,§,# Henrik Hansson,⊥ Michael E. Himmel,∥
Mats Sandgren,⊥ Jerry Ståhlberg,⊥ and Gregg T. Beckham*,‡
†
Department of Chemical and Materials Engineering and Center for Computational Sciences, University of Kentucky, 177 F. Paul
Anderson Tower, Lexington, Kentucky 40506, United States
‡
National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United
States
§
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208,
United States
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

∥
Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States
⊥
Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala BioCenter, Almas allé 5,
Downloaded via UNIV FED DO PARANA on August 10, 2022 at 15:57:58 (UTC).

SE-75651 Uppsala, Sweden

4.3. T. reesei Cellulases: Understanding the
Mechanisms of Action 1322
4.3.1. T. reesei as an Early Model for Cellulase
Action 1322
4.3.2. GHs and Related Enzymes 1324
5. Carbohydrate-Binding Modules and Linkers 1326
5.1. Family 1 Carbohydrate-Binding Modules 1326
5.2. Linkers 1331
6. Family 7 Glycoside Hydrolases 1338
6.1. Structural Studies and Catalytic Function 1341
6.1.1. TrCel7A: Wild-Type 1341
6.1.2. TrCel7A Catalytic Mutants 1341
CONTENTS 6.1.3. F. oxysporum Cel7B with Active Site-
1. Introduction 1309 Spanning Nonhydrolyzable Inhibitor 1341
2. Cellulose 1311 6.1.4. TrCel7B 1342
2.1. Cellulose Structures 1312 6.1.5. H. insolens Cel7B S37W/P39W Mutant 1343
2.2. Cellulose Microfibrils 1314 6.1.6. TrCel7A: Cello-Oligomer Complexes 1345
2.3. Cellulose Substrates 1315 6.1.7. P. chrysosporium Cel7D 1345
2.3.1. Microcrystalline Cellulose (MCC) from 6.1.8. TrCel7A: Exo Loop Engineering 1345
Plants 1315 6.1.9. TeCel7A 1346
2.3.2. MCC from Microbes 1315 6.1.10. PcCel7D Bound with Disaccharides 1346
2.3.3. Phosphoric Acid Swollen Cellulose 6.1.11. M. albomyces Cel7B 1346
(PASC) 1316 6.1.12. Heterobasidion irregulare Cel7A 1346
2.3.4. Cellulose Crystallinity 1316 6.1.13. T. harzianum Cel7A 1347
3. Glycoside Hydrolase Catalytic Mechanisms 1316 6.1.14. L. quadripunctata Cel7B 1347
3.1. Retaining and Inverting Mechanisms 1316 6.1.15. TrCel7A Michaelis Complex and Glyco-
3.2. Carbohydrate Ring Puckering 1318 syl-Enzyme Intermediate 1347
4. Early Developments in Fungal Cellulases 1320 6.1.16. GH7 Catalytic Insights from Molecular
4.1. History of the Discovery and Improvement Simulation 1347
of T. reesei Strains: Premolecular Era 1320 6.2. Processivity, Kinetic Modeling, and Visual-
4.1.1. Early Work at U.S. Army Natick Labo- ization 1350
ratories 1320 6.3. Product Inhibition 1357
4.1.2. Early International Effort for Strain 6.4. Pyroglutamate 1359
Improvement 1320 6.5. Glycosylation 1360
4.2. New Understanding from the Molecular Era: 6.6. Protein Engineering 1361
Cloning in T. reesei and S. cerevisiae 1321 6.7. Conclusions 1362
4.2.1. Cloning and Protein Production in T. 7. Family 6 Glycoside Hydrolases 1364
reesei 1321 7.1. Structural Studies 1366
4.2.2. RUT C-30 Revealed 1322
4.2.3. Cloning T. reesei Genes in S. cerevisiae:
The Road to Consolidated Bioprocessing 1322 Received: July 2, 2014
Published: January 28, 2015

© 2015 American Chemical Society 1308 DOI: 10.1021/cr500351c

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7.1.1. TrCel6A: Wild-Type 1366 11.1. Initial Discoveries of Oxidative Function 1417
7.1.2. T. f usca Cel6A 1367 11.2. Mechanistic and Structural Studies 1419
7.1.3. TrCel6A: Y169F Variant 1367 11.3. Conclusions 1424
7.1.4. H. insolens Cel6A: Apo 1367 12. Modeling Enzymatic Hydrolysis 1425
7.1.5. H. insolens Cel6A: Cello-Oligomer Com- 12.1. Ordinary Differential Equation-Based Mod-
plexes 1368 els 1425
7.1.6. TrCel6A: Non-Hydrolyzable Ligands 1368 12.2. Agent-Based Models 1427
7.1.7. H. insolens Cel6B 1369 13. Concluding Remarks 1428
7.1.8. H. insolens Cel6A: D416A/Thio-Oligosac- Author Information 1429
charide Complex 1369 Corresponding Author 1429
7.1.9. TrCel6A: D175A and D221A Variants 1370 Author Contributions 1429
7.1.10. H. insolens Cel6A: D405N Variant 1371 Notes 1429
7.1.11. H. insolens Cel6A: D416A/Isofagomine Biographies 1429
Complex 1372 Acknowledgments 1431
7.1.12. C. cinerea Cel6C 1373 Abbreviations 1431
7.1.13. C. cinerea Cel6A 1373 References 1431
7.1.14. C. thermophilum Cel6A 1374
7.1.15. TrCel6A Variants HJPlus and 3C6P 1374
7.2. Catalytic Function 1374 1. INTRODUCTION
7.2.1. Catalytic Acid 1374 Lignocellulosic biomass has enormous potential to contribute to
7.2.2. pKa Modifying Residues 1374 worldwide energy, chemical, and material demands in a
7.2.3. Catalytic Base 1375 renewable, sustainable manner. In the United States alone, it
7.2.4. Catalytic Priming of Ring Distortion 1376 has been estimated that 30% of the current petroleum usage
7.2.5. Substrate Binding 1376 could be offset via biomass conversion to transportation fuels.1
7.2.6. Product Inhibition 1377 Indeed, in both the United States and European Union, biomass
7.2.7. Processive Catalytic Cycle 1378 is currently the most abundant source of energy from renewable
7.2.8. Synergistic and Processive Function 1378 sources. Given escalating energy demands worldwide, especially
7.3. Glycosylation 1379 in liquid transportation fuels,2,3 coupled with concerns of global
7.4. Protein Engineering 1380 climate change through continued use of fossil fuel resources, it
7.5. Conclusions 1382 is likely that biomass utilization will be a primary contributor in
8. Family 5 Glycoside Hydrolases 1383 the near- to mid-term to the global sustainable energy
8.1. Structural Studies 1384 portfolio.4−7
8.1.1. Catalytic Function 1384 All plant cells exhibit thick cell walls that primarily consist of
8.1.2. T. aurantiacus Cel5A 1386 polysaccharides and the aromatic polymer lignin. These
8.1.3. P. rhizinflata EglA/CelA 1389 polymers have evolved to form complex composite materials
8.1.4. T. reesei Cel5A (Formerly EG II/EG III) 1389 that render plant cells highly resistant to attack from pathogens.8
8.2. Characterization of Activity and Specificity 1390 Plant polysaccharides primarily consist of cellulose, the β-1,4
8.2.1. T. viride EG III 1391 linked homopolymer of glucose; hemicellulose, a heteroge-
8.2.2. TrCel5A 1391 neous, branched polysaccharide primarily made up of a β-1,4
8.2.3. H. insolens Cel5A 1393 linked polymers including xylan, glucoronxylan, xyloglucan,
8.2.4. Other Fungal GH5s 1396 glucomannan, and arabinoxylan backbones with heterogeneous
8.3. Protein Engineering 1396 side chains;9 and pectin, a typically minor component in cell
8.4. Conclusions 1397 walls consisting of a complex set of polysaccharide polymers
9. Family 12 Glycoside Hydrolases 1400 enriched in α-linked galacturonic acid or galacturonic acid and
9.1. Structural Studies 1401 rhamnose monomers.10,11 Lignin is a heterogeneous, branched,
9.1.1. Overall Structure 1402 alkyl-aromatic polymer comprising three phenyl-propanoid
9.1.2. GH12 Ligand Complex Structures 1402 monomers linked by myriad C−O and C−C bonds that are
9.2. Plant Cell Wall Loosening/Extension Activity likely formed through radical coupling reactions during cell wall
by GH12 Enzymes 1403 synthesis.12 The enzymatic machinery for the synthesis of plant
9.3. GH Clan-C: Structure and Sequence Compar- cell walls is a matter of intense research with many outstanding
ison 1404 questions.8−15 Cellulose, hemicellulose, and lignin represent
9.4. Enzyme Discovery and Engineering 1404 approximately 20−50%, 15−35%, and 10−30% of plant cell
9.5. Conclusions 1405 walls, respectively, on a dry weight basis.16 Due to its prevalence
10. Family 45 Glycoside Hydrolases 1406 as the most abundant polymer in terrestrial plants, cellulose is
10.1. Structural Studies 1409 the most abundant biological material on Earth. In addition,
10.1.1. Subfamily A 1409 cellulose is the most recalcitrant carbohydrate polymer to
10.1.2. Subfamily B 1410 catalytic degradation when compared to other plant cell wall
10.1.3. Subfamily C 1411 polysaccharides.6 Cellulose serves a key structural function in
10.2. Catalytic Function 1411 plants and is synthesized by complex enzymatic machinery
10.3. Similarities of GH45s to Expansins and during cell wall synthesis.14,15 The sugars covalently locked in
Swollenins 1411 cellulose and hemicellulose represent a vast, renewable
10.4. Conclusions 1415 feedstock for the production of fuels and chemicals. Lignin, in
11. Lytic Polysaccharide Monooxygenases 1416 addition, represents a potential feedstock for valorization to
1309 DOI: 10.1021/cr500351c
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Figure 1. Overall view of a conventional biochemical conversion process to produce fuels and chemicals from lignocellulosic biomass. Cellulase
enzymes can be used to convert the cellulose portion of nonfood biomass, such as agricultural waste and energy crops, into fermentable sugars for
subsequent conversion to renewable fuels and chemicals.
fuels and chemicals although selective conversion of lignin to
value-added products remains a significant challenge.17−19
For production of fuels and chemicals from lignocellulosic
biomass, overcoming the heterogeneity and recalcitrance of
plant cell walls in a cost-effective manner at scales sufficient to
offset fossil-fuel-derived resources is a major technical challenge.
Second-generation biofuels production facilities are currently
under development, and several have been constructed to date
around the world, with a primary aim to convert lignocellulosic
biomass to ethanol. These facilities generally utilize a
“biochemical conversion” process wherein biomass is first size
reduced through milling or chipping, followed by a mild
thermochemical pretreatment step to render plant cell wall
materials more amenable to attack by biocatalysts. The
enzymatic hydrolysis step then depolymerizes cellulose and
residual hemicellulose to sugars. The last step upgrades and
converts sugars to fuel or chemicals (Figure 1).20 In the large
suite of process options, the biomass depolymerization steps,
namely pretreatment and enzymatic hydrolysis, have long been
identified as the most costly portion of the conversion
process.21−23 Many different pretreatment options have been
examined including dilute acid, hot water, steam explosion,
ammonia fiber expansion, alkaline, lime, maleic acid, and others,
as extensively discussed in recent reviews and comprehensive
technology comparisons.24−30 The enzymatic hydrolysis step
represents the second portion of biomass depolymerization and
is a major cost driver in bioethanol production due to the high
cost of enzyme production.21−23,31 However, cellulolytic
enzymes are incredibly selective for glucose production and
produce fewer downstream catalyst inhibitors relative to high
temperature deconstruction. Thus, significant efforts have been
expended to understand and improve natural paradigms for
enzymatic depolymerization of biomass with the aim to decrease
1310
Review
the cost of sugar production for fuels and chemicals
production.6,32−34
Recently, cellulase enzyme research has been accelerated due
to renewed interest in the production of ethanol from
lignocellulosic biomass. Ethanol is a useful blend stock for
light-duty vehicles in the transportation sector, but there are
significant issues with its large-scale use including problems
associated with hygroscopy, blending limits with gasoline, and
the need for a new distribution system beyond petroleum-
derived fuels. Thus, third-generation biofuels are now under-
going focused research and development with the goal of cost-
effective production of infrastructure-compatible fuels from
lignocellulosic biomass including fuels to fulfill demands in the
gasoline, diesel, jet fuel, and maritime sectors. From the
perspective of biochemical conversion processes that utilize
carbohydrate intermediates such as clean sugars and carbohy-
drate derivatives as feedstocks, this body of work can be very
broadly categorized into biological and catalytic upgrading of
sugars to hydrocarbon fuels.5,35−40 Multiple strategies have
emerged for each class of fuel and chemical production, which
have been widely reviewed in the past several years.5,35−40 Many
of these strategies still rely on the production of sugars or sugar
derivatives, and thus, there remains significant incentive to
reduce the cost of selective biomass depolymerization to
carbohydrates through some combination of thermochemical
pretreatment and enzymatic hydrolysis.
Many organisms across the kingdoms of life have evolved the
necessary enzymatic machinery for converting cellulose to
soluble species for a food and energy source. Given the
complexity of plant cell walls, most biomass-degrading
organisms employ a battery of enzymes with synergistic function
to break down polysaccharides41,42 and, in some cases,
lignin.43−47 To date, two primary paradigms have been
discovered in cellulose depolymerization: the “free” enzyme
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Chemical Reviews
paradigm and the cellulosomal paradigm. The free enzyme
paradigm represents the case wherein enzymes diffuse as single
catalytic units, often accompanied by binding modules
covalently attached together via linker domains, exemplified
by the enzyme suite from the filamentous fungus Trichoderma
reesei (or Hypocrea jecorina).41 The primary mode of action of
free cellulases is one wherein endoglucanases (EGs), or
nonprocessive cellulases, act by cleaving cellulose chains in
amorphous regions, and cellobiohydrolases (CBHs), or
processive cellulases, attach to cellulose chain ends and
depolymerize and hydrolyze cellulose chains typically into
disaccharide units, down the length of a chain. In solution,
cellobiose is then cleaved by β-glucosidases into glucose for
cellular uptake by organisms. The recent discovery of selective,
oxidative enzymes adds another element of endo-acting action
to this paradigm wherein chains are likely cleaved in crystalline
regions.48−52
The other well-characterized paradigm for enzymatic biomass
degradation is the use of cellulosomes wherein noncovalent
cohesin−dockerin interactions enable large complexes up to
hundreds of enzymes to operate in close proximity on large
protein scaffolds. Cellulosomes were first discovered in the
anaerobic rumen bacterium, Clostridium thermocellum.53−56
More recently, additional paradigms have perhaps begun to
emerge57,58 including one with multimodular enzymes that
contain multiple catalytic domains (CDs) per protein, which
appears to be a natural “interpolation” between free cellulases
and cellulosomes in function,59 and another with polysaccharide
utilization loci, but additional characterization of these potential
new paradigms will be required.
Of all biomass-degrading organisms, fungi play a pivotal role,
as they are responsible for the vast majority the biomass
degradation in nature. Fungi have colonized a vast range of
terrestrial and marine environments, and are thus of vital
importance for the recycling of carbon on Earth, a capacity with
broad implications in global ecology, biogeochemistry,
agriculture, and, more recently, for industrial applications.
Broadly, two primary modes of fungal biomass degradation exist
employing either enzymatic or chemical means to break down
biomass. Brown-rot fungi initially utilize Fenton chemistry to
generate hydroxyl radicals, which attack plant cell walls via
powerful oxidation reactions.60,61 Conversely, filamentous fungi
characterized as soft-rots and white-rots have been long known
to primarily employ enzymatic means to break down biomass.
Many filamentous fungi produce high titers of effective
cellulolytic enzymes employing the “free” enzyme paradigm
mentioned above, and thus, filamentous fungal enzyme
production has become a cornerstone of industrial biofuels
research and development.6,34 In particular, the isolation of the
soft-rot ascomycete fungus T. reesei in the South Pacific in the
1940s followed by its characterization at the Natick Research
Laboratories marked the beginning of the development of
filamentous fungi for biomass conversion purposes.62 Sub-
sequently, a wealth of mechanistic information regarding fungal
cellulase structure and function has been reported from the
1980s onward. This research area has been accelerated in the
past decade by significant governmental and commercial
investments into research into biofuels production worldwide.
Here, we review enzymatic mechanisms utilized by fungi to
depolymerize cellulose, with the primary focus on discoveries
made since the first structural reports for each enzyme family.
We highlight open questions related to furthering our
understanding of these biologically and industrially important
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Review
enzymes. Cellulases, unlike many commonly studied enzymes,
act on an insoluble substrate, and cellulose represents a complex,
heterogeneous macromolecule; thus, we first briefly review
physical and chemical aspects of cellulose relevant to enzyme−
substrate interactions. The initial structural reports of cellulases
primarily beginning in the early 1990s enabled identification of
substrate interactions and catalytic mechanisms, and given that
most fungal enzymes that depolymerize cellulose employ a
hydrolytic mechanism, we discuss general aspects of how
cellulases prime cellulose for hydrolysis. We then briefly review
research efforts into cellulase mechanisms up until the initial
structural reports. Many well-characterized cellulases are
multimodular with binding modules and linkers that enable
multiple functions within the same enzyme; the efforts
dedicated to the study of binding function and multimodularity
are reviewed here as well. As fungi and most other biomass-
degrading organisms secrete enzyme cocktails, we then discuss
the primary components of fungal cellulolytic cocktails
including glycoside hydrolase (GH) family 7, 6, 5, 12, and 45
cellulases, in this order. We note that we do not discuss β-
glucosidases (GH1 and GH3 enzymes), which cleave cellobiose
to glucose in solution as these enzymes have been recently
reviewed and primarily act on soluble substrates.63 Additionally,
fungi and other biomass-degrading organisms employ a vast
diversity of enzymes aimed at other substrates in the plant cell
wall such as hemicelluloses and pectins; these polysaccharides
are also not reviewed here, primarily because cellulose is the
most recalcitrant carbohydrate substrate in the cell wall
compared to the other polysaccharides, but the importance of
other cell wall polysaccharides is of significant relevance for
biomass conversion. For each GH family, we focus on the
molecular-level aspects of cellulase action by reviewing studies
from structural biology, biophysical and biochemical measure-
ments including microscopy, spectroscopy, scattering, and
modeling. All of these tools are needed to develop a
comprehensive, mechanistic view of cellulase structure and
function. Additionally, we review exciting new discoveries in
mechanistic paradigms for cellulose depolymerization, namely
the recent discovery of lytic polysaccharide monooxygenases
(LPMOs), which employ copper, oxygen, and a reducing agent
to oxidatively cleave cellulose. In summary, work in recent years
has demonstrated that substantial gains are still possible in
reducing enzyme loadings for biomass conversion, and for
further gains, fundamental science, enzyme discovery and
screening, and improved methods for enzyme engineering will
be required.
2. CELLULOSE
Cellulose is the β-1,4-linked homopolymer of β-D-glucose and
exhibits a reducing and nonreducing end, the former of which
can ring open to produce an aldehyde form. Given its natural
prevalence and utility as a fuel and chemical precursor, and as a
material with myriad applications, the study of cellulose is vast
and diverse. As such, we briefly review salient physical and
chemical properties relevant to cellulose deconstruction by
cellulolytic enzymes in nature. Even this narrowing of scope
leaves a great deal of informative and influential work to be
addressed. We note this section is by no means meant to be an
exhaustive review of cellulose structure, but, rather, a brief
introduction setting the stage for discussion of cellulose
deconstruction and the methods used to study cellulases. Lastly,
we note that the mechanisms of cellulose synthesis, which are
key for elucidating its structure in plants, have long been studied,
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Figure 2. Natural and synthetic cellulose polymorphs. For each of the four polymorphs, the “end-on” view is shown at top and the “top-down” view at
bottom. Celluloses Iα and Iβ are naturally occurring polymorphs exhibiting only intralayer hydrogen bonding.88,89 The differences between the two
polymorphs are most easily observed from the “top down” view, which illustrates the subtle differences in interlayer chain stacking. Celluloses II and
IIII, the result of chemically pretreating cellulose I, are significantly different in their chain stacking arrangement.90,94 Hydrogen bonding, shown in
yellow, occurs between sheets as across the layers.

and identified as one of the major challenges in plant
biology.14,64−66 Exciting recent work has begun to illustrate
the molecular level details of cellulose biosynthesis.15,67−70
1312
2.1. Cellulose Structures
Enzymes that break down recalcitrant polysaccharides must
overcome multiple challenges in their catalytic action. At the
atomic level, β-1,4-linked polysaccharides exhibit incredibly
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Figure 3. Four of the putative Iβ microfibril models proposed in recent years. For many years, the 36-chain diamond shape has been the prevailing
model.64,104−108 NMR and X-ray scattering techniques have suggested alternative models may better fit the data. Fernandes et al. suggested the 24-
chain rectangle and diamond models, the former of which was also proposed by Thomas et al.102,103 Newman et al. recently suggested the 18-chain
diamond models fits wide-angle X-ray scattering data well.109
strong covalent bonds. In an influential study, Wolfenden and
colleagues estimated that the uncatalyzed half-life of O-
glycosidic linkages such as those found in cellulose, chitin, and
other polysaccharides are 2 and 4 orders of magnitude more
stable than DNA or peptide bonds, respectively, to uncatalyzed
hydrolysis at neutral pH with a half-life of an astounding 5
million years.71,72 Indeed, chemically intact cellulose and chitin
(a similar substrate based on N-acetylglucosamine monomers)
have been found in fossilized plants that are significantly
older.73−77 Given these bond strengths, GHs are incredibly
proficient enzymes in that they can provide rate enhancements
(kcat/kon) up to 1017-fold. This in turn makes GHs the most
powerful hydrolytic enzymes known to man that do not employ
metals or other cofactors.78,79
The covalent bond strength of the β-1,4 glycosidic linkage in
cellulose is merely one of the challenges that enzymes face when
depolymerizing this recalcitrant substrate. Cellulose microfibrils
in plants pack into tightly bound, crystalline lattices wherein
only a fraction of the chains are accessible to enzymatic attack on
the microfibril surface, which forms yet another barrier that
fungi and other biomass-degrading organisms must overcome.
Individual cellulose chains are cosynthesized by large,
membrane-bound terminal complexes, which simultaneously
extrude and assemble multiple chains of cellulose into
elementary microfibrils.14,64−66 Understanding how enzymes
depolymerize cellulose from a mechanistic perspective is
predicated on knowing the localized crystalline structure of
cellulose chains and the shapes and properties of the cellulose
microfibrils. Each question is briefly described below.
Cellulose can pack into multiple crystalline forms, or
polymorphs. Natural systems, including plants, produce
cellulose I, the study of which dates back to the 19th
century.80−84 The structure of cellulose I as a set of parallel-
1313
Review
oriented chains was originally reported by Gardner and
Blackwell in 1974 using cellulose from the algae Valonia
ventricosa.85 In 1984, plant cellulose was further shown to be a
mixture of the cellulose Iβ and Iα polymorphs in two papers
from Vanderhart and Atalla.86,87 In 2002 and 2003, Nishiyama
and co-workers reported refined structures of both cellulose Iβ
and Iα obtained from a tunicate (Halocynthia roretzi) and from
the freshwater algae Glaucocystis nostochinearum.88,89 These
structures were obtained from synchrotron X-ray and neutron
diffraction studies of oriented cellulose films and were obtained
at nearly atomic resolution in both cases such that hydrogen
bond patterns could be established. The primary differences
between the cellulose Iβ and Iα polymorphs reside in the
hydrogen bonding patterns and the interlayer chain stacking
arrangement (Figure 2). Cellulose Iβ forms two different layers,
dubbed the “center” and “origin” layers, whereas cellulose Iα
forms a unit cell with a single chain. In both cellulose I
polymorphs, the hydrogen bonds only exist within single layers
with no intersheet hydrogen bonds, which was noted at the time
to be surprising, further suggesting that the van der Waals
contacts in cellulose contribute a great deal to its overall
thermodynamic stability. It should also be noted that both
cellulose structures originated from cellulose microfibrils that
are known to be much bigger in diameter than elementary plant
cellulose microfibrils, which may have an impact on the
structures, as discussed in more detail below.
Certain chemical treatments can convert cellulose I to other
crystalline forms. Treatment with solvents such as sodium
hydroxide (known as mercerization)90 or dissolution in ionic
liquids91,92 can convert the parallel chains in native cellulose into
an antiparallel arrangement with both inter- and intralayer
hydrogen bonding interactions, producing cellulose II (Figure
2). The structure of cellulose II was also recently presented.90,93
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Figure 4. Computer simulation suggests that the cellulose Iβ microfibril twists by approximately 1.5° per cellobiose unit when it is solvated in water
and the ends are not fixed. The existence of cellulose twist remains an open experimental question. A representative microfibril begins to twist after
only 1 ns, shown at left. Reprinted with permission from ref 114. Copyright 2005 Elsevier Ltd.
Less severe chemical treatments, for example in ammonia, can
also convert cellulose I or cellulose II into cellulose III
(sometimes called cellulose IIII and IIIII indicating that it
originates from cellulose I or II, respectively).94 Cellulose III
forms staggered layers with intra- and interlayer hydrogen
bonding interactions, unlike in cellulose I where the chains pack
into flat layers (Figure 2). Both cellulose II and III typically
exhibit greater digestibility by cellulase enzymes. 20,95,96
Cellulose IV has been suggested as another polymorph,97 but
less characterization has been conducted to date, and it has been
recently suggested that it is likely quite similar to cellulose Iβ.98
Several thermodynamic measurements have been conducted in
the 1980s to understand the differences between cellulose I and
II, which revealed relative enthalpic stabilities;99 additional
rigorous thermodynamic and kinetic experiments will be
required to more fully understand the interconversion between
cellulose polymorphs.
2.2. Cellulose Microfibrils
In the cellulose Iβ and Iα structures determined from Nishiyama
et al.,88,89 the cellulose microfibrils were very large in diameter
(10−20 nm).88,89 Conversely, plant cellulose chains pack into
microfibrils with much smaller diameters,100−103 and, thus, have
much higher surface-to-volume ratios than cellulose from algae
or tunicates. Regardless of the source, the cross-sectional shape
of the microfibril is quite likely dictated by the shape and
arrangement of the terminal synthase complexes. It has long
been hypothesized that the elementary microfibril size in plants
is 36 chains, primarily on the basis of imaging of terminal
complexes combined with other analyses (Figure 3).64,104−108
More recently, new reports have begun to challenge the notion
of a 36-chain model by applying a variety of scattering and
nuclear magnetic resonance (NMR) techniques.100,102,103,109
Kennedy and co-workers examined cellulose from celery
collenchyma, which is similar to cellulose from higher plants,
using NMR and scattering methods. In a careful study with their
assumptions discussed thoroughly, they conclude that the
microfibril diameters are between 2.4 and 3.2 nm, which
1314
Review
corresponds to a cellulose microfibril consisting of 15−25 chains
each. In 2011, the same group used a wide variety of scattering
and spectroscopic tools to examine microfibril cross sections in
spruce wood.102 With the assumption that the number of chains
in the microfibril must be divisible by 6, their findings suggest
that spruce wood microfibrils comprise about 24 chains. In
terms of microfibril shape, they suggest either a diamond or a
rectangular shaped microfibril, with the latter model fitting their
experimental observations better (Figure 3). Moreover, their
results suggest that the microfibrils are likely twisted and that the
surfaces are disordered.102 In 2013, the same group again
examined celery collenchyma cellulose microfibrils and
demonstrated that the best-fit model was a 24-chain microfibril
with 8 layers of 3 chains each.103 Subsequently, a study from
Newman et al. applied similar methods to mung bean
cellulose.109 They fit their scattering and NMR data to a 36-
chain microfibril model but were not able to match the
experimental data to the calculated diffractograms. Conversely,
Newman et al. were able to achieve good agreement between
the computed and measured diffractograms and spectra using a
24-chain and 18-chain model, with the 18-chain model
providing the best fit (Figure 3). Overall, this recent body of
work suggests that cellulose microfibrils in higher plants may be
smaller than the commonly assumed 36-chain models.
Computer simulations have also been applied alongside of
structural studies to gain additional insights into molecular level
cellulose properties.110−113 Simulations in particular have
provided supporting evidence for the twisting of microfibrils,
and as such, several recent highlights that are complementary to
structural, NMR, and scattering studies are discussed here. In an
early study, Matthews et al. simulated a 36-chain model of
cellulose Iβ and demonstrated that the microfibril is prone to
twisting by approximately 1.5° per cellobiose unit (Figure 4).114
Additional computational studies have reported twisting in
microfibrils of various sizes and shapes with multiple empirically
fit energetic representations (force fields) for the carbohy-
drates,115−118 and recent simulations have suggested the
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physical basis for this twisting phenomenon.119 As mentioned
above, experimental studies have also suggested that plant
microfibrils indeed exhibit twist.102 The effects of cellulose
microfibril twisting on cellulase action largely remain unan-
swered.
Beyond atomistic simulations, multiple groups have devel-
oped coarse-grained models for studying cellulose at increas-
ingly larger scales and at a variety of resolutions.120−127 Coarse-
grained models are quite important for the study of cellulose
phenomenon such as enzyme action on the substrate,
interactions of multiple cellulose microfibrils, or interactions
with other biopolymers, all of which occur at long length and
time scales. Going forward, improved simulation models for
cellulose coupled to the development of high-resolution imaging
will likely converge such that simulation and experiment can be
directly connected. To this end, Ciesielski et al. recently
reported the development of a transmission electron micros-
copy (TEM) method wherein atomic coordinates for cellulose
microfibrils can be mapped onto the experimentally measured
nanoscale architecture of the plant cell wall after mild
thermochemical pretreatment.128 This study highlights the
power of combined experimental and computational tools to
understand the behavior of cellulose microfibrils in the cell wall
context, which is key to understanding the physical and chemical
environments that cellulases encounter during their enzymatic
action.
It is commonly thought that cellulose comprises crystalline
and amorphous regions. Although significant research has been
conducted toward this question, there remains much con-
troversy around this topic, and a clear definition of amorphous
cellulose does not exist. Habibi et al. note that amorphous
cellulose likely arises from chain dislocations wherein micro-
fibrils distort due to internal strain.129 Taking the definition of
amorphous cellulose as distorted regions along the length of
crystalline fibers aligns well with the conventional model of
cellulose hydrolysis by GH enzyme cocktails32 mentioned
previously. Namely, EGs cleave cellulose chains in amorphous
regions along the cellulose crystals, and CBHs attach to chains
and processively hydrolyze glycosidic linkages down the chain.
During processive hydrolysis in crystalline regions, CBHs must
decrystallize individual chains of cellulose, which requires
thermodynamic work. Several studies have examined the
amount of work that is required to decrystallize chains from
the surface of cellulose microfibrils with computer simula-
tion.130−132 Beckham et al. examined chain decrystallization of
cellulose Iβ, Iα, II, and III demonstrating that the work to
decrystallize chains of celluloses Iβ and Iα is greater than that of
celluloses II and III.130 Moreover, it was shown that an
increasing number of inter- and intralayer interactions increase
the decrystallization work in essentially an equivalent manner,
further suggesting that interlayer and intralayer interactions
contribute similarly to the thermodynamic stabilization of
cellulose.130
2.3. Cellulose Substrates
Accurate experimental representation of the nano- and micro-
structures of cellulose is a notoriously difficult prospect.
Throughout the cellulase literature, multiple model celluloses
are used as representative crystalline and amorphous substrates.
Commonly, Avicel, bacterial microcrystalline cellulose
(BMCC), tunicate or algal cellulose, various forms of pretreated
biomass, or non-natural polymorphs derived from the many
types of cellulose I are used to represent crystalline cellulose.
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Amorphous cellulose is often represented by phosphoric acid
swollen cellulose (PASC), regenerated cellulose, or soluble,
oligomeric substrates. Each of the “clean” cellulose substrates
(i.e., free of hemicellulose or lignin) exhibit significantly
different properties including degree of polymerization, degree
of crystallinity, microfibrils and fiber shapes, available reactive
surface area for cellulase reactions, and possibly more variables
that substantially impact the effectiveness of enzymatic
deconstruction. The use of many model substrates, often with
significant variation even within substrates, can make direct
comparison of cellulase performance data difficult across
laboratories and studies. For pretreated substrates, which
typically contain other residual plant cell wall polymers, direct
comparisons are even more challenging. Thus, we note that
caution should be taken when comparing activity data across
studies as substrate variations can greatly influence the outcomes
of activity measurements. As the study of cellulase action is
invariably linked the substrate properties, these properties
should not be overlooked. Detailed substrate characterization is
essential in cellulase studies. Going forward, the combination of
structural, nanoscale imaging, and modeling will continue to be
important for elucidating the features of cellulose relevant to
enzymatic deconstruction. Some of the more prevalent
substrates are described below.
2.3.1. Microcrystalline Cellulose (MCC) from Plants.
Purified, microcrystalline celluloses have been used extensively
for comparing cellulose performance.96 These celluloses are
made from wood pulp and are available commercially, including
the following: Sigmacell, Solka-floc, and Avicel. Cotton linters,
the dust-like byproduct from cotton processing, are also used for
cellulose assays. Cotton linters are essentially fragmented, but
chemically unmodified cotton fibers reduced to small size (ca.
100 mesh). Whatman No. 1 filter paper, commonly used to
determine the international filter paper unit, is made from
cotton cellulose. The amorphous cellulose content of MCC
(Avicel) can be increased by a mechanical treatment process
known as ball milling. Ball-milled cellulose, especially when
prepared under conditions of low water content, had been
shown to be considerably more digestible than the starting
material.127,133
2.3.2. MCC from Microbes. Cellulose is produced by green
algae and some bacteria, primarily of the genera Acetobacter,
Sarcina, and Agrobacterium. Acetobacter xylinum cellulose is most
commonly used for cellulose digestion experiments as it can be
produced in fermenters in yields as high as 15 g/L.134 The
bacterial cell produces protofibrils of approximately 2−4 nm in
diameter, which are eventually bundled into ribbon-shaped
microfibrils of about 80 × 4 nm2.135 Natural bacterial cellulose
(BC) must be purified before use in enzyme assays. For A.
xylinum cellulose, cellulose from culture medium is treated with
sodium or potassium hydroxide, acetic acid, followed by
repeated washing with ultrapure water.136 The resulting
BMCC has microfibrils of approximately 0.1−10 μm in width,
which is 100 times thinner than the microfibrils found in plant
cell walls. It has also been demonstrated that purified A. xylinum
BMCC has a degree of polymerization of about 800.137 Green
algae in which crystalline cellulose is the major component of
the cell walls include the Cladophorales (Cladophora, Chaeto-
morpha, Rhizoclonium, and Microdyction) and a few members of
Siphonocladales (Valonia, Dictyosphaeria, Siphonocladus, and
Boergesenia). V. ventricosa produces large cellulose microfibrils
compared to plant cell wall microfibrils),138 which have been
proposed to be in a 33 × 38 chain configuration with lengths of
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hundreds of nanometers to a few micrometers.139 Valonia and
Cladophora produce celluloses with an exceptionally high degree
of crystallinity, approximately 95% from XRD,140 and for this
reason make ideal substrates for cellulase studies. Algal cellulose
is indeed complex, perhaps more than plant cellulose,
considering that, in each Cladophora microfibril, the two
cellulose I polymorphs were suggested to coexist, alternating
either longitudinally or laterally.141 Koyama et al. further
suggested that there are three types of cellulose I polymorphs
found in green algae: Iα-broad microfibrils, Iβ-flat ribbons, and
Iβ-small microfibrils with random orientation.142 Although
highly crystalline, the specific surface area of Cladophora
cellulose powder has been reported to be as high as 95 m2/g
from N2 gas adsorption studies,143 which is much higher than
this value for BMCC, ∼1 m2/g. Today, the molecular and
polymeric basis for the action of cellulases on algal celluloses is
not clear. The advantages of using BMCC and algal celluloses
are thus considerable, yet inconsistencies in preparation
practices between laboratories can introduce difficulties in
comparing assay results.
2.3.3. Phosphoric Acid Swollen Cellulose (PASC).
Walseth first developed a procedure for producing high-
reactivity cellulose suitable for cellulose activity studies by
swelling air-dried cellulose in 85% phosphoric acid.144 After
dissolving crystalline cellulose, the solubilized precursors can be
formed, which can subsequently be hydrolyzed. Cellulose
dissolution in phosphoric acid involves two processes: an
esterification reaction between hydroxyl groups of cellulose and
phosphoric acid to form cellulose phosphate and a competition
of hydrogen bond formation between the hydroxyl groups of
cellulose and hydrogen bond formation between hydroxyl
groups of cellulose and water molecules or hydrogen ions.145
During this acid treatment, some glycosidic bond hydrolysis
occurs which reduces the degree of polymerization, although the
effects can be controlled. Following regeneration with water,
free phosphoric acid is recovered, and the resulting cellulose is
amorphous without significant recrystallization. PASC has been
used widely as a test substrate for CBHs. Note that, in contrast
to PASC, which has no chemical modification, carboxymethyl
cellulose (CMC) retains esterified carboxymethyl side chains
and is thus suitable only for testing EG action.
2.3.4. Cellulose Crystallinity. The crystallinity index (CI)
of celluloses is a key parameter when selecting substrates for
enzyme assays. Cellulose CI has been measured using several
different techniques including XRD, solid-state 13C NMR,
infrared (IR) spectroscopy, and Raman spectroscopy.146 There
have also been several methods used for calculating CI from the
raw spectrographic data, particularly for XRD. Methods using
Fourier transform (FT)-IR spectroscopy determine CI by
measuring relative peak heights or areas.147,148 Thygesen et al.
compared four different analysis techniques involving XRD and
reported that the CI of Avicel cellulose varied significantly
depending on the technique used.149 In 2012, Park et al. made
critical comparisons between the different techniques using
XRD and solid-state 13C NMR.150 Comparisons were made with
literature data for the CI of one type of cellulose (Avicel PH-
101) using these methods. Park et al. also reported the CI values
for seven crystalline cellulose preparations and BMCC, and
further recommended that the simple, peak height XRD
measurement be excluded from comparisons and the remaining
values from other XRD and NMR methods be averaged to
obtain the “best value” for CI. These results are shown in Table
1.
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Review
Table 1. Values for CI from Combined XRD and NMR
Analysis150
cellulose XRD peak XRD amorphous NMR C4 peak
tested deconvolution subtraction separation average
Cladophora ND 80 ND 80
BMCC 73 82 74 76
Avicel PH- 61 78 57 65
101
SigmaCell 50 61 79 56 65
SigmaCell 20 64 67 53 61
SolkaFloc 57 57 44 53
3. GLYCOSIDE HYDROLASE CATALYTIC
MECHANISMS
Given the diversity of monosaccharides and the multiple types
of glycosidic linkages possible, carbohydrates form the most
diverse set of biomolecules in nature. As such, the enzymatic
machinery to synthesize, modify, and deconstruct carbohydrates
is vast.151−153 The Carbohydrate-Active Enzymes Database
(www.CAZy.org) is a manually curated list of the primary
enzyme classes known to act on carbohydrates.151−153 Since its
inception in 1998, CAZy has become an invaluable resource in
carbohydrate enzymology. More recently, a sister site,
CAZypedia (www.cazypedia.org), has begun to develop
descriptions of the CAZy Database entries for each enzyme
class and family within each class. The protein classes covered in
CAZy as of the time of this review include glycosyltransferases,
carbohydrate esterases, polysaccharide lyases, auxiliary activities,
carbohydrate-binding modules (CBMs), and GHs. Glycosyl-
transferases (EC 2.4-) catalyze the formation of glycosidic bonds
with nucleotide phosphate or lipid phosphate leaving groups
and to date have been classified into 95 families. 154
Carbohydrate esterases (EC 3.1.1- or 3.1.5-) are responsible
for de-O- or de-N-acylation of polysaccharides, such as acetyl
xylan esterases, and comprise 16 known families with multiple
nonclassified sequences, perhaps suggesting that other families
exist. Polysaccharide lyases (EC 4.2.2-)155 employ β-elimination
reaction mechanisms to cleave uronic acid-contaning poly-
saccharides, such as those commonly found in pectins.10,11 To
date, polysaccharide lyases form 23 characterized families.151,155
“Auxiliary activities” or AAs are a more recent addition to
CAZy,152 and currently include 12 families of enzymes in total,
with 8 known to be active during lignin degradation and 4
known to be directly active on polysaccharides, namely LPMOs.
Oftentimes, catalytic function for carbohydrates is associated
with binding function, and thus, CAZy contains a classification
scheme for CBMs as well, which to date represent 69 families, as
will be discussed in section 5.156 Lastly, GHs are included in the
CAZy Database. GHs are the primary drivers of enzymatic
polysaccharide degradation in nature and represent a vast set of
enzymes. To date, 132 GH families have been characterized. As
most cellulases are GHs, we focus on their description in this
section from a general, mechanistic viewpoint. Individual GH
families that fungi employ, namely GHs from Families 5, 6, 7,
12, and 45, are described in separate sections below. We note
that newly discovered LPMOs do not employ a hydrolytic
mechanism; their mechanism of action is described in section
11.
3.1. Retaining and Inverting Mechanisms
As proposed by Koshland in 1953, nearly all known GHs
employ one of two mechanisms: either retaining or inverting
hydrolysis.157 Inverting mechanisms proceed via a single
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Scheme 1. Two Primary Catalytic Mechanisms of GHsa

a
(A) Inverting GHs employs a single displacement catalytic mechanism wherein a water molecule conducts nucleophilic attack at the anomeric
carbon of the −1 sugar, a catalytic base abstracts a proton from the attacking water molecule, and a catalytic acid transfers a proton to the glycosidic
oxygen to cleave the glycosidic linkage, resulting in an inversion of stereochemistry at the anomeric carbon. (B) Retaining GHs employs a two-step,
double displacement catalytic mechanism. In the first step, the nucleophilic residue attacks the anomeric carbon simultaneously with the proton
transfer from the acid residue to the glycosidic oxygen resulting in the formation of the glycosyl-enzyme intermediate and cleavage of the glycosidic
bond. In the second step, a water molecule enters the active site and attacks the anomeric carbon simultaneously transferring a proton to the catalytic
base, thus restoring the enzyme active site for subsequent catalysis.

catalytic step (Scheme 1A), wherein a water molecule conducts
nucleophilic attack at the anomeric carbon of an oligosaccharide
or polysaccharide, a proton from water is transferred to the
catalytic base, and a proton is transferred from the catalytic acid
to cleave the glycosidic linkage. Generally, both the catalytic acid
and base residues exhibit carboxylate groups (i.e., Asp or Glu).
This reaction results in an inversion of stereochemistry at the
anomeric center. Before the next catalytic cycle, the catalytic acid
and base must be reset to their configuration for catalysis.
Scheme 1A illustrates the inversion from a β-linkage to an α-
linkage. Conversely, retaining mechanisms are two-step
reactions (Scheme 1B). The first step in retaining hydrolysis
(typically termed “glycosylation”) involves proton transfer from
the catalytic acid and attack at the anomeric carbon by the
nucleophile residue to form a covalent glycosyl-enzyme
intermediate and invert the stereochemistry of the sugar
covalently bound to the enzyme. In the second step, termed
“deglycosylation”, a water molecule enters the enzyme active site
and conducts nucleophilic attack at the anomeric carbon. The
glycosyl-enzyme intermediate bond is broken, and a proton is
transferred to the catalytic base (which was the acid in the first
step) resetting the enzyme and again inverting the stereocenter
at the anomeric carbon for an overall net retention of
stereochemistry. In retaining mechanisms, the carbohydrate
product of glycosylation is typically not assumed or illustrated to
participate in deglycosylation, and generally, both the catalytic
acid and nucleophile are carboxylate residues. Several GH
families have been discovered that do not follow this typical
paradigm, which have been recently reviewed.158 All cellulases
reviewed here follow the typical retaining or inverting hydrolysis
mechanisms shown in Scheme 1 with the exception of LPMOs
reviewed in section 11.
1317
While the mechanisms put forth by Koshland are now widely
accepted,159−161 there has historically been some debate about
whether bond cleavage and formation occurs in concerted steps
in an SN2 reaction, as shown in Scheme 1, or via a carbocation
intermediate as in an SN1-type reaction,162−166 or even via an
acyclic oxocarbenium ion,167 although subsequent studies have
not supported ring-opening as part of the mechanism.168
Researchers now discuss oxocarbenium-like transition states
(TSs), rather than a distinct ionic intermediate, as the lifetime of
an ion in an enzyme active site would be less than a molecular
vibration.169−171 The oxocarbenium-like TSs show extended
distances between atoms involved in bond cleavage and
formation, sometimes referred to as “exploded” TS.161,170
GH enzymes often have multiple carbohydrate binding sites
in their CDs. For example, family 7 glycoside hydrolase (GH7)
cellulases exhibit at least 9 subsites for binding cello-oligomers,
and catalysis generally takes place 2 glucose units from the
reducing end of the chain bound in the enzyme tunnel.172−174
As GHs ubiquitously feature binding subsites for carbohydrate
residues in polysaccharides, Davies, Wilson, and Henrissat
proposed a scheme for the naming of carbohydrate binding sites
similar to the scheme previously proposed by Biely, Krátký, and
Vršanská.175,176 Specifically, they propose the use of the −n to
+n system used by molecular enzymologists, where −n
represents the nonreducing end and the +n represents the
reducing end subsites. Thus, glycosidic bond cleavage in GHs
occurs between the −1 and +1 subsites. This nomenclature has
become nearly universally accepted in the carbohydrate
enzymology and structural biology communities and will be
used throughout this review.175,176
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Figure 5. Representations of the IUPAC conformations of six-membered rings: chair (C) has four atoms in the same plane, with one atom above that
plane and an atom on the opposite side of the ring below the plane; envelope (E) has five atoms in one plane, with the sixth either above or below the
plane; half-chair (H) has four atoms in one plane, with one atom above the plane and an adjacent atom below the plane; skew (S) has four atoms in the
same plane, with one atom above the plane and an atom two positions away below the plane; and boat (B) has four atoms in the same plane, with two
atoms on opposite sides of the ring either both above or both below the plane.
3.2. Carbohydrate Ring Puckering
A significant number of crystallographic studies have been
conducted to date to study GH catalysis, as detailed in
subsequent sections of this review for cellulolytic enzymes,
and many studies have employed transition state analogues to
capture the Michaelis complex of carbohydrates in GH active
sites. A universal observation to date is that GHs distort
carbohydrate ring geometries in Michaelis complexes for
catalysis away from the chair conformations that are
thermodynamically stable in aqueous solution.159,161,169,177,178
To systematically classify sugar puckering geometries, Schwartz
developed the original nomenclature for describing the 38
canonical puckering conformations of pyranose rings,179 which
was subsequently adopted by the International Union of Pure
and Applied Chemistry (IUPAC).180 This system describes
pyranose rings as chair (C), envelope (E), half-chair (H), skew
(S), and boat (B) conformations, as shown in Figure 5. The
naming system further uses superscript and subscript numbers
for each ring conformation to denote which atoms are outside of
the reference plane formed by four atoms (e.g., B1,4 denotes a
boat-shaped pyranose ring with the C1 and C4 carbons below
the reference plane). To quantitatively describe puckering,
Cremer and Pople proposed a spherical coordinate system that
uniquely describes the pyranose ring conformations as a
function of three parameters that describe a sphere (Figure
6).181 This has become the standard method to quantify the
Figure 6. 38 IUPAC designated puckering conformations for six-
membered rings are projected here on a two-dimension representation
of the Cremer−Pople sphere.
degree of ring puckering and is quite useful when stable
puckering geometries in enzymes are intermediate between the
38 IUPAC puckering geometries.182−184 Hill and Reilly
introduced a system of triangular decomposition that is
particularly well-suited for molecular simulations and also
uniquely identifies the exact puckering conformation.185
Carbohydrate ring puckering in GH active sites has been
identified since the seminal structural study of the hen egg-white
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Review
lysozyme,186 which was the first structure of an enzyme ever
solved.187 On the basis of analogy with SN1 transition states, it
was suggested that the ring distortion is a key component of the
hydrolysis mechanism, contributing to the formation of a
carbonium ion and weakening the C1-glycosidic bond.186 Other
research groups have proposed that the role of ring distortion is
overestimated on the basis of lack of difference in observed
binding constants188 or calculated low-energy conformations.189
However, structural studies of the hen egg-white lysozyme
confirmed that a key substrate ring is distorted from the chair
conformation, which investigators attributed to forming a
geometry that supported formation of an oxocarbenium ion at
C1190 or to weaken the scissile glycosidic bond by creating a
higher-energy ground state.162,191
In one of the first published structures of a cellulase,
specifically a family 6 glycoside hydrolase (GH6), Rouvinen et
al. noted that the sugar ring in the −1 subsite (at the time
referred to as the “B subsite”) was distorted from the solution-
stable 4C1 conformation. The authors were originally unsure if
this distortion was functionally significant.192 Barr et al. later
reported that GH mutants that could allow the −1 sugar to relax
to a chair conformation exhibit increasing binding affinity and
decrease hydrolytic activity, supporting the proposal that ring
distortion is important for catalysis.193 Zou et al. observed
distorted sugars in the −1 subsite studies of T. reesei Cel6A
(TrCel6A) with a nonhydrolyzable ligand, identifying the
residue whose steric clash with the hydroxymethyl group forces
the sugar ring into a distorted conformation.194 They proposed
that the distortion is integral to the catalytic mechanism,
providing for nonperiplanar orientation between the scissile
bond and a doubly occupied, nonbonding orbital from the ring
oxygen.194 As reviewed in detail below, many more structures
were solved of cellulases that have revealed puckered
carbohydrate rings in the −1 subsites; certainly, the same is
also true for GHs beyond cellulolytic GHs, but these additional
structures are outside of the scope of this review. For capturing
such catalytic reaction coordinates, GH structural biology efforts
have relied on the use of synthetic ligands, such as thio-linked
sugars, which cannot be hydrolyzed, or with other transition
state analogue ligands in native enzymes.161,178,195−197 Withers
et al. pioneered the use of fluoro-sugars in mechanistic GH
studies wherein fluorine atoms are substituted for hydroxyl
groups in pyranose sugars. For example, substitution at the C2
position can result in a decrease in deglycosylation rates in
retaining GHs, enabling capture of glycosyl-enzyme intermedi-
ates.198−202 Other substitutions can lead to the ability to probe
mechanistic steps in GHs when coupled to NMR, mass
spectrometry, and enzyme kinetics analysis.203 Moreover, the
use of native substrates in catalytically inactive mutants, such as
the mutation of glutamate, a common acid/base to glutamine is
a common approach. Through approximately a decade of work
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starting in the 1990s, it became clear that cellulases from a
variety of GH families commonly distort pyranose rings from
the 4C1 conformation to a puckered conformation, observed in
the 1S3,204−208 2SO,192,194,209 BO,3,192,209 or 2,5B210 geometries.
The preponderance of data indicating conservation of puckering
in enzymes from a wide range of GH families and different
organisms indicates that ring distortion indeed plays a central
role in catalysis of glycosidic bond cleavage, and spurred efforts
to further understand the role of puckering in catalysis.
Proposals for the role of puckering include providing for
antiperiplanar alignment of the ring oxygen lone pair with the
leaving group (the scissile glycosidic bond), a requirement of
Deslongchamps’s theory of stereoelectronic control,211 later
referred by the more specific name “antiperiplanar lone pair
hypothesis (ALPH)”.212 This requirement in enzymatic
reactions has been refuted on the basis that predictions from
this theory have been proven false,169,170 such as the prediction
that α-linked substrates would retain their ground state, chair
conformation during reaction.166,213 In counterpoint to such
objections, Nerinckx et al. noted that observed pucker
conformations seemingly counter to the ALPH could in fact
substantiate the ALPH if they were points on a catalytic itinerary
with the 1C4 inverted chair, rather than the solution-stable 4C1
chair conformation, and the 1C4 geometry could convert to 4C1
in a subsequent step.214 This suggestion opposes the “principle
of least nuclear motion” which posits that enzymatic itineraries
adopt conformations which minimize such large conformational
changes.168
Deslongchamps further theorized that ring “distortion raises
the energy of the ground state and thus lowers the energy of
activation for bond cleavage”,166 in agreement with the earlier
proposal by Jencks.162 Warshel vigorously repudiated this “strain
theory” based on theoretical models showing that it cannot
provide a significant catalytic effect.215,216 Additionally,
researchers have noted that observed puckering conformations
aligned β-linkages in an axial position suitable for nucleophilic
attack.159,186,217,218 While it may be a factor in catalysis, it is not
a universal feature of GH substrate puckered geometries.184
Warshel advocated that the most important enzymatic
contribution to catalysis is stereoelectronic stabilization of the
transition state,215,216 harkening back to Pauling’s theories on
enzyme mechanisms.219 Blake et al. originally proposed a
stereoelectronic argument for ring distortion, suggesting that the
puckered conformation aids catalysis by allowing the ring
oxygen to share charge with the anomeric carbon, stabilizing a
carbonium ion formed during the proposed reaction mecha-
nism.163,186 As previously discussed, it is now widely believed
that GHs employ oxocarbenium-like TSs, rather than ionic
intermediates, but the basic concept of the puckered geometry
stabilizing a positive charge at the anomeric carbon still applies,
and stabilizing the TS is exactly what Pauling and Warshel
champion. The proposal that puckered ring geometries stabilize
positive charge at the anomeric carbon continues to hold wide
support.159,161,196,220
As modifications to the electronic structure of carbohydrate
ring puckering is now an obvious feature of GH mechanisms,
quantum mechanical approaches coupled to structural biology
studies are a clear means to probe the mechanistic under-
pinnings of carbohydrate catalysis.159 A series of theoretical
studies have been conducted to determine the effects of ring
distortion on catalysis. Smith performed one of the earlier
studies using 2-oxanol.221 This work suggested that ring
distortion may obviate the need to form a high-energy
1319
Review
oxocarboniom ion (instead forming a lower-energy oxonium
ion−water complex) as well as lowering the transition state
energy by stretching the scissile glycosidic bond.221 To
investigate whether preferential properties for catalysis could
be observed in isolated puckered monosaccharides, several
studies have examined differences between puckered con-
formations of β-D-glucose. The metadynamics studies by Biarnés
et al. investigated the relative stability of different puckered
conformations by employing Cartesian-collective variables to
explore the puckering potential energy surface.222 While the
chosen collective variables resulted in distortions in puckering
amplitude and energy barriers,223 Biarnés et al. found that bond
lengths and partial charges of key atoms change as a function of
puckering geometry, with puckered geometries observed in
enzyme complexes displaying catalytically favorable properties
such as elongated C1−O1 bond distance, shortened C1−O5
bond distance, and a higher partial charge on C1.222
Barnett and Naidoo performed a study of β-D-glucose
puckering ensuring more complete sampling of puckering
geometries, revealing the free-energy landscape calculated with
the semiempirical method PM3CARB-1.182 The initial inves-
tigation was followed by a study in which they compared free
energy differences calculated with different semiempirical
methods against the density functional theory method
B3LYP/6-311++G(d,p).183 The trends revealed by the semi-
empircal methods were qualitatively similar to notable
quantitative differences: PM3CARB-1 revealed that the
second-lowest energy conformation is BO,3, just 1.6 kcal/mol
higher in free energy than the lowest-energy 4C1 conformation
obtained, while B3LYP/6-311++G(d,p) identified BO,3 as the
third-lowest conformation, at 5.5 kcal/mol higher than lowest-
energy 4C1 conformation. Together, these papers confirm that
different puckered geometries afford measurably different
properties that would make them more or less amenable to
catalysis, and they show the results are sensitive to the method
employed.
Recently, Mayes et al. employed a highly accurate electronic
structure method (CCSD(T)/6-311+G(d,p)//B3LYP/6-
311+G(2df,p)) in a study that ensured thorough sampling of
monosaccharide ring geometries.184 In addition to the 38
IUPAC puckered conformations, monosaccharides have
exocylic groups that are free to rotate at ambient temperatures,
resulting in the notorious carbohydrate flexibility that challenges
structural and dynamic properties of carbohydrates.224 The
study by Mayes et al. compared puckering behavior of five
biologically important sugars, and the differences among them
testify to the importance of exocyclic groups in defining
puckering landscapes. For β-D-glucose, they confirmed that the
puckered conformations employed by GH active sites offer a
combination of catalytically advantageous properties, such as a
higher partial charge at C1 to make it a better target for
nucleophilic attack. Furthermore, these conformations have
lower barriers for ring interconversion.
In the following sections, we review specific details of each
cellulase family represented prevalently in fungi, which comprise
both inverting and retaining enzymes. Attention is given
primarily to specific elements of the catalytic steps in each
cellulase family. For more comprehensive reviews of general GH
catalytic mechanisms, we refer readers to recent re-
views.158,159,161,196
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Figure 7. Geneaology of T. reesei mutants developed internationally from World War II to about 2000. Mutagenesis was affected by radiation or
chemical treatments. At the time, exposure to a linear accelerator was an effective means of irradiation. Most commercial preparations used today are
based on proprietary improvements of RUT-C30.
4. EARLY DEVELOPMENTS IN FUNGAL CELLULASES
Prior to the landmark publications of the first structures of
fungal cellulases, many biochemical experiments shed light on
the nature of cellulase enzymes, especially from the model
fungus T. reesei. As a preface to detailed structural and
mechanistic descriptions, we first briefly describe some of the
initial developments in cellulase enzymology related to the
initial isolation of T. reesei, the early development work to
produce mutant strains for industrial production of cellulases,
and the first studies related to fingerprinting the cellulolytic
cocktail of T. reesei. We note that the naming scheme of
cellulases changed from their initial characterization to adopt the
standard CAZy GH classification. Throughout section 4, we
typically employ the “classical” name [e.g., the previously used
T. reesei “CBH I” versus the new, commonly used T. reesei
“Cel7A” (TrCel7A)], and provide the new, widely used enzyme
names at the end of this section in Table 2. Beyond this section,
we only utilize the new naming schemes for enzymes.
4.1. History of the Discovery and Improvement of T. reesei
Strains: Premolecular Era
4.1.1. Early Work at U.S. Army Natick Laboratories. The
history of the discovery and improvement in the Trichoderma
strains has been thoroughly studied and reviewed.225−229 Many
Trichoderma species are known today to be cellulolytic, a
characteristic of their saprophytic life style. These strains include
T. reesei, T. lignorum, T. koningii, T. harzianum, T. long-
ibrachiatrum, T. virens, and T. pseudokoningii. Studies of
decomposing cotton militaria sent from Bougainville Island
(Solomon Islands) in the South Pacific to the U.S. Army
Quarter Master Research and Development Center at Natick,
Massachusetts, forged our understanding of an important
cellulolytic fungus, eventually named T. viride QM6a.62 Note
that T. viride was eventually reclassified as T. reesei in honor of
Elwyn Reese.6 Postwar work at Natick by Reese and Mandels
led to the modern concept of saccharification of biomass to
fermentable sugars affected by the powerful T. reesei enzyme
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cocktails.230 An important aside is that, later in the 1970s, Gauss
and co-workers at the Bio Research Center Company (Nagoya,
Japan) patented the concept of combining, in one tank, T. reesei
fungal enzymes, milled biomass, and fermentative organisms.231
This revolutionary concept, later improved in a U.S. patent
owned by the Gulf Research & Development Company,232
dramatically increased thermodynamic “pull” to products, and
was called simultaneous saccharification and fermentation, or
SSF.
The wild-type strain of T. reesei, QM6a, has been thoroughly
studied and, importantly, used to generate the modern strains of
enhanced industrial microorganisms by radiation mutagenesis.
T. reesei QM9414, generated from QM9123, was the first
production grade strain (Figure 7).233 QM9414 was found to
produce approximately 2 times more cellulase protein than
QM9123,234 which was in turn superior to the QM6a parent
strain by about the same extent.235 This era of very early strain
improvement is today difficult to follow for a number of reasons,
including the limited methods of protein and activity
determination available at the time. For example, the Biuret
copper oxidation assay, known today to be highly influenced by
sugars and amino acids, was the dominant protein assay. Today,
the bicinchoninic acid or dinitrosalicylic acid reagent assays are
preferred.236 The other problem was that workers worldwide
used vastly different cellulase performance assays, normally
based on lab-specific digestion curves, given simply as “units”. A
true universal cellulase assay, first proposed by Ghose in 1987,
helped alleviate these problems by defining a relevant measure
for cellulases, later known as the international filter paper
unit.237
4.1.2. Early International Effort for Strain Improve-
ment. Starting from QM9414, researchers at the Natick Lab
used ultraviolet light to generate the MCB-77 series of
mutants.238 MCB-77 strains demonstrated volumetric produc-
tivity of 90 IU/L/h, compared to 30 IU/L/h for QM9414.239
The M series of mutants, produced at Rutgers University from
QM9414, yielded strain M-7.240 M-7 was then treated with
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nitrosoguanidine, which, after suitable performance selection on
cellulose, resulted in the NG T. reesei mutant series. Strain NG-
14 showed approximately 5 times the filter paper activity, and
twice the activities of cellobiase (or β-glucosidase) and EG of
QM9414.241 NG-14 also produced about twice as much
cellulase protein as QM9414, or about 1.4 mg/mL.
Montenecourt and Eveleigh used T. reesei NG-14 to generate
the next series of mutants by chemical mutagenesis, including
the C and E series. From this series, the Rutgers C30 and E58
are of interest as they are carbon catabolite derepressed strains.
Both of these strains showed about 4−6 times the cellobiase (or
β-glucosidase) activity of QM6a, although the filter paper
activity was comparable. The RUT-C30 strain was superior to all
other strains reported at the time, producing about 2.2 g/L
cellulase protein,240 a trait later attributed to an increased
endoplasmic reticulum content.242 Application of RUT-C30 to
SSF further enhanced its performance on cellulose and
biomass.243 Another hypercellulolytic strain produced during
the late 1970s at Rutgers was RL-P37.240 RL-P37 is thought to
be the parent strain further developed by the U.S. cellulase
industry and used for large-scale production today. During this
time, reports of a Cetus Corporation propriety strain, known as
L27, a regulatory mutant of QM9414, revealed that considerable
effort was being applied to improving T. reesei by the industrial
sector.244 Besides the cellulase improvement programs dis-
cussed above at Natick and Rutgers, industrial sponsorship of
this work was also ongoing in the 1980s−1990s in Finland
(VTT), the United States (Cetus), Japan (Kyowa), and France
(CAYLA).233 Smaller programs were also underway in Portugal,
Czechoslovakia, and India. A report from Portnoy (2011)
suggested that the only other T. reesei strain roughly comparable
to RUT-C30, CAYLA’s CL-847, was in fact derived from RUT-
C30.245
4.2. New Understanding from the Molecular Era: Cloning in
T. reesei and S. cerevisiae
4.2.1. Cloning and Protein Production in T. reesei. In
1987, Penttilä et al. reported the successful transformation of T.
reesei using a plasmid carrying the dominant selectable marker
amdS.246 Heterologous DNA was found to be integrated at
several different locations in the T. reesei genome, often in
multiple tandem copies. The successful expression of an
Escherichia coli β-galactosidase in T. reesei was also reported by
these workers.246 This same year, the same group further
reported the characterization of the major genes coding
cellulases from T. reesei.247 The group from VTT reported the
first review of homologous and heterologous protein expression
in T. reesei, citing both promoter changes and gene inactivation
as critical for successful outcomes.248 The strong, inducible
promotor for the cbh1 gene is highly recommended in this
review. In the same year, Harkki et al.249 reported the first
example of genetic engineering of T. reesei for the purpose of
varying the production of key cellulases.249 These workers
produced a strain of T. reesei in which the cbh1 gene was
inactivated and the gene coding the major EG, egl1, was cloned
into a vector carrying the CBH I promotor and terminator,
which resulted in the overexpression of EGI. The following year,
1992, progress to date and outlook for cloning heterologous
genes in T. reesei were discussed in a landmark review from
VTT.250 The ability to utilize a CBH I delete strain of T. reesei
was demonstrated the following year by the publication of
perhaps the first paper reporting the engineering of this enzyme.
Srisodsuk et al. showed that the linker of the three domain T.
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reesei CBH I [CD−linker−cellulose binding domain (CBD)]
could be shortened or otherwise modified genetically to explore
its possible role in cellulose saccharification, as discussed in
section 5.251
At this time, new tools were proposed for improving the
transformation efficiency of T. reesei, notably hygromycin
resistance.252 In 1995, Nakari−Setälä et al. reported the
development of a modified strain of T. reesei able to grow on
glucose, where catabolic repression shuts down the normal
cascade of cellulase expression, and produces targeted proteins
using novel promotors.253 Although expression levels of the
cellulase CDs reported in this study were low, about 100 mg/L,
this report marked a very important early step in engineering
promotor performance as well as movement toward engineered
T. reesei strains that were able to secrete heterologous genes into
the culture medium without the high background interference
from other cellulases. A critical review in 1995 by Keränen and
Penttilä outlined the state of the art for expression of
heterologous proteins in filamentous fungi.254 The next year, a
report from Ilmén et al. described the isolation and character-
ization of the cre1 genes of the filamentous fungi T. reesei and T.
harzianum.255 We know today that, in multicellular ascomycetes,
the C2H2 type transcription factor CreA/CRE1 acts as a
repressor mediating carbon catabolite repression.256 In T. reesei,
CreA/CRE1 binds to the promoters of the respective target
genes via the consensus motif, 5′-SYGGRG-3′, and inhibits
translation.257 Ilmén et al.258 also reported the first detailed
analyses of the CBH I promotor. This work set the stage for
follow-up work from the same group the next year which began
to describe regulation of cellulase expression in T. reesei at the
molecular level.259 In 2000, Pakula et al. reported the
development of a novel isotope labeling approach for measuring
protein synthesis and secretion in T. reesei in an attempt to
understand the rate-limited events occurring in cellulase
production.260 Over the next few years, work at VTT continued
to investigate the protein transcription/expression factors
responsible for efficient production of cellulases, including
ACEI, ACEII, and RHOIII.261−263 A few years later, the VTT
group reported the characterization of two genes, ire1 and ptc2,
implicated in T. reesei unfolded protein response.264
Following this early work, reports began to emerge regarding
the development of new strains of T. reesei that had been
genetically modified to relieve catabolite repression and thus
improve target protein production when cultures are grown on
glucose. A leading example was the report from VTT
summarizing development of strains of T. reesei in which the
cre1 genes were deleted or modified by truncation (cre1-I) to
improve expression of plant cell wall degrading enzymes under
noninducing growth conditions.265 It is noteworthy that the
cre1-I gene was found earlier in the RUT C30 strain and thus
explains some of the ability of this strain, produced by chemical
mutagenesis in the 1980s, to produce cellulases when grown on
glucose. Kubicek et al.266 summarized promising genetic
strategies for improving protein production in T. reesei. It is
known that plant cell wall degrading enzymes are inducible and
under both positive and negative transcriptional control. In this
review, the authors summarize what is known about the known
positive control elements include XYR1, ACE2, and HAP2/3/5,
and the negative elements are ACE1 and CRE1. The authors
further propose increased research focus on signal transduction
pathways and other potential factors known to influence gene
regulation, such as light cycling and intensity. More recently,
Steiger et al.267 reported the next generation of T. reesei stains
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developed for engineering, namely strains that are susceptible to
homologous gene integration and employ reusable bidirection-
ally selectable markers.
4.2.2. RUT C-30 Revealed. In the modern era of systems
biology, the critical mutations conferring superior performance
to RUT-C30 and its descendants have been elucidated.268 In
2009, a massively parallel sequencing effort was used to compare
the genomes of T. reesei RUT-C30 and its direct ancestor NG14
with the published genome of the wild-type organism, QM6a.269
The genomes of RUT-C30 and NG14 were found to be missing
over 100 kb of genomic DNA present in QM6a, encompassing
18 large deletions in RUT-C30. These deletions included the
cre1 gene truncation identified earlier by Ilmén et al.258 and 15
small deletions or insertions in RUT-C30. As stated above, cre1
regulates catabolite repression in most cellulolytic fungi and is
responsible for the strong inhibitory effect glucose has on
cellulase production.258,270 For wild-type Trichoderma, the
“cellulase signaling cascade” is initiated by natural environ-
mental chemical inducers, such as cellobiose. However, large-
scale enzyme production is usually conducted with more
powerful inducers, such as sophorose and lactose. We note that
a process has recently been reported which converts glucose
rich, biomass derived monosaccharide mixtures to sophorose
and other products using enzymatic transglycosylation,271
providing a more cost-effective source of this inducer. Beyond
the mutations to cre1, 211 single nucleotide variants in RUT-
C30 were found by Ilmén et al.258 and Seidl et al.270 These
mutations and deletions affected 43 genes in NG14 and in RUT-
C30.269 One of these genes in RUT-C30, gls2α, encodes the
glucosidase II α-subunit,272 an enzyme important for trimming
N-linked oligosaccharides in glycoproteins, such as cellulases.
Indeed, detailed biochemical analyses of Cel7A purified from
RUT-C30 broth shows evidence that this enzyme is not
glycosylated normally.273 As noted by Peterson and Nevalai-
nen,274 several of the industrial strains derived from QM6a were
selected for resistance to the chemical mutagen, nitro-
soguanidine, which is now known to be a glycosylation
inhibitor.275
In contrast to QM6a, RUT-C30 can be grown on glucose for
cellulase production, and improvements in growing and
inducing RUT-C30 have led to reports of its cellulase
productivity as high as 30 g/L. Proprietary strains of T. reesei
used in industry, based on RUT-C30 and its descendants, have
led to strains delivering more than 100 g/L cellulase protein.229
In spite of this outstanding industrial record, RUT-C30 remains
a marginal producer of heterologous proteins. Examples of such
expression results include the following, in order of highest
production: Hormoconis resinae glucoamylase P (0.5 g/L),276
Melanocarpus albomyces laccase (0.23 g/L),277 Acrophialophora
nainiana XynVI (0.17 g/L),278 and CBH I-Fab fusion antibody
(0.15 g/L).279 All other examples of protein expression from
RUT-C30 reported are below these levels. However, T. reesei
strain ALKO3620 (produced from QM9414 by multiple rounds
of mutation) was reported to produce 1.9 g/L of Nonomuraea
f lexuosa Xyn11A,280 which may make this case the highest
publicly available heterologous protein expression example in a
mutant T. reesei strain. Peterson and Nevalainen274 suggest that
the causes of poor heterologous protein expression lie in the
incompatibilities of foreign peptides with natural mechanisms of
protein recognition and disposal within the cell, known
collectively as the unfolded protein response.
4.2.3. Cloning T. reesei Genes in S. cerevisiae: The Road
to Consolidated Bioprocessing. In many ways, the concept
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of direct microbial conversion, also known as consolidated
bioprocessing, inspired the early work in T. reesei molecular
biology. The molecular cloning era of T. reesei cellulases began
with simultaneous reports from the U.S. and Europe in the
October 1983 issue of Bio/Technology that the T. reesei gene,
cbh1, had been successfully cloned in the heterologous host, E.
coli, using lambda phage technology.281 The work from North
America was reported by the research team from Cetus
Corporation,281 and the work from Europe was reported by
VTT.282 At this time, it was the stated goal of Cetus Corporation
to transform S. cerevisiae with the four dominant cellulase genes
from T. reesei in order to engineer a cellulose digesting, ethanol
producing organism. To demonstrate the level of international
competition ongoing at this time, we note that four year later
workers at VTT reported several landmark accomplishments
relevant to this same objective. First, Penttilä et al. reported the
successful cloning of active EG I and EG III in S. cerevisiae using
the yeast phosphoglycerate kinase promoter.283 At this time,
these workers noted that the molecular weight of EGIII was
higher than expected and proposed potential problems with
hyper-glycosylation in yeast. Importantly, in 1988, Penttilä et al.
reported the expression of the CBH I and CBH II in S.
cerevisiae.284 However, these heterologous enzymes were found
to be highly polydisperse in molecular weight, to bind poorly to
crystalline cellulose, and to be active only on amorphous
cellulose. Pilot scale (200 L) production of T. reesei CBH II in S.
cerevisiae was reported by VTT in 1990.285 Cetus Corporation
discontinued its work on cellulases in the mid-1980s and was
sold to Chiron Corporation in 1991. In 1993, this group
reported the coexpression of CBH II and EG I in S. cerevisiae and
showed that both enzymes were active on crystalline cellulose
and acted synergistically.286 The group at VTT continued to
express T. reesei genes in S. cerevisiae well into the next decade,
for example, including expression of the family 5 glycoside
hydrolase (GH5) mannanase gene, man1.287 The next leap in
understanding of heterologous gene processing in S. cerevisiae
resulted from studies of the unfolded protein response.288
Many years later, work to express T. reesei plant cell wall
degrading enzymes continues worldwide, and VTT has
remained involved in this field. In 2011, a notable report
describes a systematic study to express, at higher titers than
previously reported, active CBH I and II in S. cerevisiae using an
engineered chimera approach.289 These authors were able to
report the fermentation of MCC to ethanol by S. cerevisiae
strains expressing CBHs with the addition of β-glucosidase. This
concept is, of course, the direct microbial conversion or
consolidated bioprocessing process. The team authoring this
work included authors from VTT, Mascoma Corporation, and
the University of Stellenbosch, South Africa. In the year of this
review, Voutilainen et al. have reported the expression of active,
thermal stable chimeric CBHs in S. cerevisiae.290
4.3. T. reesei Cellulases: Understanding the Mechanisms of
Action
4.3.1. T. reesei as an Early Model for Cellulase Action.
Likely because of the significant resource availability for T. reesei
at the time, this fungus soon became the archetype micro-
organism used to study cellulase digestion at the level of the
enzymes it produces, instead of the World War II era focus on
microbial action and effects. In 1950, Reese and co-workers
reported that although many fungi could hydrolyze derivatized
celluloses, only a few could grow on crystalline cellulose, such as
cotton.291 At this time, the molecular nature of the cellulase
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concept was not known. We note that, during the war years,
only crude salt and metal ion mediated solvent precipitation of
proteins could be applied to protein separations. These
methods, developed for fractionating blood plasma proteins
for the war effort, were entirely unsuitable for purification of
microbial enzymes. This and other postwar developments in
protein biochemistry, erupting into play immediately after the
war, helped the nascent cellulase biochemistry programs.
Perhaps the most significant of these developments was the
work in the 1950s on enhanced protein separation methodology
in Uppsala, Sweden. Work by Pederson in Uppsala during this
era led to the development of modern size exclusion
chromatography with early columns being packed with simple
agarose gel spheres.292 With the ability to separate active,
secreted enzymes, the advent of modern cellulase biochemistry
was born.
Simple, atmospheric pressure column chromatographic
techniques were used in the early and mid-1960s to purify
cellulases for study.293−295 As this was before the time of
widespread use of electrophoretic gels, analytical ultracentrifu-
gation was the primary tool for characterizing the molecular
weights and degree of polydispersity of proteins. Using these
new tools for biochemistry, Mandels and Reese293 suggested a
protein level concept for cellulase action, called “C1−Cx”
(Figure 8). Enabled by this newly acquired ability to study the
Figure 8. Diagram of first concept for the roles and specificities of
enzymes hydrolyzing cellulose, known as the C1−Cx model. Adapted
with permission from ref 293. Copyright 1964 Society for Industrial
Microbiology and 1999 Springer Science and Business Media.
action of relatively homogeneous enzyme preparations and
measure their respective concentrations accurately, they
described in this report the possible role of C1 enzymes,
required for attack on crystalline cellulose, and the more
prevalent Cx enzymes, needed for hydrolysis of soluble,
derivatized cellulose such as PASC. Characterization of the
hypothetical C1 enzyme lagged behind progress on the Cx
enzymes for many years. Putative Cx enzymes were identified as
endo-(1−4)-β-glucanases, exo-(1−4)-β-glucanases, and β-gluco-
sidases.296 During the 1960s, it was commonly thought that the
C1 and Cx enzymes worked in a synergistic way to hydrolyze
crystalline cellulose, although no molecular detail was available.
One proposal during this time was that C1 was a protein that
decrystallized cellulose by displacing native hydrogen bonds in
the microfibril, leading to a more available structure for Cx.297 In
1969, Eriksson298 is credited with first proposing that an
exoglucanase could be playing the role of C1, a view supported
three years later in a report by Halliwell and co-workers.299 The
landmark work reported by Berghem and Pettersson in 1973300
clearly showed that an exo-cellulase, purified from T. viride, was
highly active on crystalline cellulose and the best candidate for
C1. This enzyme, later classified as “cellulose 1,4-β-cellobiosi-
dase (nonreducing end) or CBH I”, EC 3.2.1.91; is today
reclassified as EC 3.2.1.176 “cellulose 1,4-β-cellobiosidase
(reducing end)”.151 The picture emerging from work done by
the late-1990s is depicted in Figure 9. In this view, EGs attack
amorphous cellulose surface regions of the microfibril, revealing
new cleavage sites for exoglucanases. Exoglucanases also attack
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Figure 9. This cartoon represents the classical endo/exo model of
cellulase enzyme action in T. reesei and many other cellulolytic fungi.
Here, the dominant EG, EG I, or Cel7B acts only on amorphous
cellulose and functions as the Cx activity described by Reese decades
before.
the available free cellulose ends, with β-glucosidases converting
cellobiose to glucose. Although a second exoglucanase from T.
reesei had been reported for some time, CBH I (Cel7A) and
CBH II (Cel6A) were shown to be functionally distinct by van
Tilbeurgh and co-workers301 and to correspond to distinct
cellulose digestion morphologies determined microscopi-
cally.302 The same year, Wood proposed a model depicting
the cellulose stereochemical specificity of these enzymes, called
CBH (B′) and CBH (B), a concept not discussed much today.
In 1984, van Tilbeurgh and co-workers were first to describe the
systematic multistep chromatographic purification of all key
cellulases from T. reesei.303
A curious observation from this time was that the optimum
synergistic ratio of cellulases purified from T. reesei was CBH
I:EG I (1:1) and CBH II:EG II (95:1).305 For the latter case,
these results are consistent with the view that the EG creates
new chain ends for the exoglucanase in an almost catalytic role.
However, for the GH7 enzymes (CBH I and EG I, later known
as Cel7A and Cel7B, respectively), the 1:1 ratio suggests another
mechanism. Wood306 proposed that the CBH I/EG I pair
function with close physical coupling so that, immediately
following an internal bond cleavage event by EG I, CBH I is able
to immediately occupy the newly revealed reducing chain end.
This not only permits rapid hydrolysis, but also reduces the
possibly that the broken chain can “reanneal” into the crystal
surface. We are not aware that more recent work has confirmed
or denied this theory; however, given the challenges now known
for removing all traces of EG contamination from CBH I
preparations, caution is recommended.
Initially reported by Ståhlberg et al.304 and more recently by
Kurašin and Väljamäe307 is a view of CBH I mechanism that was
not envisioned by the simple exo/endo model. Using cellulose
reducing end group analysis following digestion, Ståhlberg et al.
suggested that T. reesei produces no true exo-cellulases (Figure
10). The work by Kurašin and Väljamäe used a similar reducing
end analysis strategy to show that both TrCel7A and
Phanerochaete chrysosporium Cel7D are also able to conduct
“endo-initiation” as well as the well-known exo-initiation leading
to hydrolysis. It is worth noting that, even with this “extra”
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Figure 10. Concept of endo-initiating CBHs was introduced by
Ståhlberg et al. in 1993.304 This new role for both CBH I or Cel7A and
CBH II or Cel6A supports somewhat the concept of C1; however, the
endo-initiation function may not be as dramatic as first envisioned by
Reese.
reactive feature, CBH I by itself cannot hydrolyze more than
about 60% of a model cellulose substrate, such as Avicel.
Today, cellulase digestions mechanisms are once again viewed
in a sense related to the original concepts from Reese, i.e., C1
and Cx. However, some inconsistencies are apparent given the
classical view shown in Figure 9. Reese suggested that C1 was a
decrystallizing protein factor, whose function may not be bond-
breaking (at least, not hydrolytic), but instead possesses an
ability to swell or disrupt cellulose, perhaps by intercalating
between cellulose chains in the elementary microfibril, the
consequence of which is action by other enzymes causing
complete digestion to cellobiose. The view shown in Figure 9
actually suggests that Cx is not a single class of enzymes, but the
combined action of EGs and exoglucanases that work together
to expose the necessary ends of cellulose chains needed to yield
simple sugars. Neither the EGs nor the exoglucanases from fungi
cause global decrystallization of cellulose. This hydrolytic
enzyme system functions in an ablative manner, peeling one
layer at a time. The essential dilemma here is that no single
protein is known to dramatically reduce cellulose crystallinity.
Some proteins may cause some local cellulose disruption and
morphological changes, for example, the expansins308 and
expansin-like proteins (swollenins),309 but their reported effects
on subsequent hydrolytic action are modest.310
So, are there protein factors that truly reflect the notions of
Reese four decades ago? The relatively recent work to define the
action of LPMOs has again posed some new potential answers
to this question. LPMOs were originally classified as fungal
GH61 enzymes and nonfungal members of family 33 CBM, but
have been reclassified,152 and are now implicated in the oxidative
cleavage of cellulose and other plant polymers. As reviewed
extensively in section 11, LPMOs are known to act in two
reaction schemes on crystalline cellulose, generating oxidized
and nonoxidized chain ends (Figure 11). Some LPMOs have
been shown to oxidize glucose at position C1, releasing lactones
that are hydrolyzed to aldonic acids,311 whereas other enzymes
act on the nonreducing end, producing ketoaldoses, or a
combination thereof.312 The copper oxidases seem to attack the
highly crystalline regions of cellulose in contrast to hydrolases.
In this regard, LPMOs may act like Reese’s C1 enzymes.313
4.3.2. GHs and Related Enzymes. Today, the secretome of
T. reesei is fairly well-understood, thanks to decades of
traditional biochemistry and the recent assembling of much of
the genome, i.e., 34 Mbp of nearly contiguous sequence
comprising 9129 predicted genes.41 T. reesei QM6a is known to
produce at least 193 GHs, 93 glycosyl transferases, 5
polysaccharide lyases, 17 carbohydrate esterases, and 41
CBMs.314 It has been pointed out that the outstanding
performance of T. reesei on biomass is somewhat surprising,
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Figure 11. Current view of cellulose degradation by many filamentous
fungi, combining both hydrolytic and oxidative fragmentation reactions.
Note that the action of LPMO may provide a new chain end for CBH I
(or Cel7A), even though it is oxidized.
considering that other fungi have a greater diversity of critical
cellulase components. For example, T. virens, Aspergillus
nidulans, and Postia placenta have 256, 251, and 248 GHs,
respectively.268 The CAZy database lists two CBHs, six EGs,
two LPMOs, and seven β-D-glucosidases for QM6a. Further-
more, as illustrated by Martinez and co-workers,41 T. reesei has
an extremely limited set of the enzymes needed to degrade plant
cell walls (accessory enzymes, hemicellulases, acetyl esterases)
and especially living walls (pectinases). Seiboth and co-
workers268 concluded that T. reesei’s success in the biosphere
must stem from its efficient cellulase induction system and
extremely high cellulase production and secretion capability.
Some caveats to this cocktail paradigm deserve attention.
First, it has been recently suggested193 that some cellulases
should be categorized as “processive EGs” for structural and
functional reasons. Structurally, some cellulases have binding
sites that are intermediate between the closed tunnels of CBHs
and the open clefts of EGs. Functionally, some enzymes have
intermediate levels of processivity, depending on the definition
of processivity used.315 Also, as discussed above, for many years,
enzymes now referred to as “cellobiohydrolases” were called
“exoglucanases” because of their assumed tendency to begin
their processive runs at a cellulose chain end (thus, “exo”).
However, the term exoglucanase is now essentially obsolete as a
class of GH given the early and now more recently confirmed
revelation that GH7 CBHs can perform hydrolysis in an endo-
initiation fashion.304,307 In addition, the more recently
discovered class of enzymes known as LPMOs (formerly
GH61 or family 33 CBM (CBM33)) have been recognized as a
vital part of an efficient cellulase cocktail. These enzymes are not
GHs, even though they were once listed in GH or CBM families.
Given these nuanced (and at times misleading) enzymatic
classifications, it may be more helpful and appropriate to think
in terms of “modes of actions” rather than types of cellulases.
As suggested above, T. reesei secretes a multiplicity of the key
cellulose degrading enzymes, especially the β-glucosidases, EGs,
and CBHs. The enzymes shown in Table 2 are those cited
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 Table 2. T. reesei QM9414 and QM6a Cellulases Reported in CAZy151−153
product con-
classical name CAZy name common name figuration reported by substrate specificity ref
Bgl2 EC3.2.1.21 GH1 Cel1A β-glucosidase 2 retaining Takashima et p-nitrophenyl-β-D-glucoside, p-nitrophenyl-β-D-cellobioside (pNPC), methylumbelliferyl-β-D- Takashima et al., 1999,317 Saloheimo et al.
al., 1999317 glucoside, 5-bromo-4-chloro-3-indolyl-β-D-glucoside 2002318
EC3.2.1.21 GH1 Cel1B β-glucosidase retaining Foreman et predicted β-glucosidase activity Foreman et al., 2003319
Chemical Reviews

al., 2003319
Bg1 EC3.2.1.21 GH3 Cel3A β-glucosidase 1 retaining Mach, Glc2/Glc3/Glc4/Glc5/Glc6, gentiobiose, laminaribiose, laminaritriose, sophorose, 2-chloro-4- Korotkova et al., 2009,321 Karkehabadi et al.,
1993320 nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside, CMC, laminarin, β- 2014322
glucan
EC3.2.1.21 GH3 Cel3B β-glucosidase retaining Foreman et predicted β-glucosidase activity Foreman et al., 2003319
al., 2003319
EC3.2.1.21 GH3 Cel3C β-glucosidase retaining Foreman et predicted β-glucosidase activity Foreman et al., 2003319
al., 2003319
EC3.2.1.21 GH3 Cel3D β-glucosidase retaining Foreman et predicted β-glucosidase activity Foreman et al., 2003319
al., 2003319
EC3.2.1.21 GH3 Cel3E β-glucosidase retaining Foreman et predicted β-glucosidase activity Foreman et al., 2003319
al., 2003319
EGII (formerly GH5 Cel5A endoglucanase II retaining Saloheimo et CMC-Na, Avicel, ball-milled cellulose, PASC Qin et al., 2008324
EGIII) al., 1988323
EC3.2.1.4
EC3.2.1.4 GH5 Cel5B endoglucanase retaining Foreman et predicted endoglucanase activity Foreman et al., 2003319
al., 2003319
CBHII GH6 Cel6A cellobiohydrolase II inverting Teeri et al., Avicel, CMC, Glc3/Glc4/Glc5/Glc6, PASC Poidevin et al., 2013,326 Ståhlberg et al., 1993304
EC3.2.1.91 1987325
CBHI GH7 Cel7A cellobiohydrolase I retaining Shoemaker et 4-methylumbelliferyl-β-D-lactoside, 2-chloro-4-nitrophenol-β-D-lactoside, 3,4-dinitrophenyl-β- Boer and Koivula, 2003,327 Becker et al., 2001328

1325
EC3.2.1.176 al., 1983281 D-cellobioside, 3,4-dinitrophenyl-β-D-lactoside, BMCC
EC3.2.1.91
EGI EC3.2.1.4 GH7 Cel7B endoglucanase I retaining Penttilä et al., Glc3/Glc4/Glc5/Glc6, pNPC, pNPL, PASC, Avicel, BC, pretreated corn stover, CMC, Van Arsdell et al., 1987,330 Biely et al., 1991,331
1986329 xyloglucan, xylan, arabinoxylan, mannan, galactomannan, barley β-glucan, hydroxyethylcellu- Bailey et al., 1999,286 Vlasenko et al., 2010332
lose
EGIII GH12 Cel12A endoglucanase III retaining Fowler et al., CMC, PASC, Avicel, Glc4/Glc5, barley β-glucan, glucomannan, filter paper Karlsson et al., 2002334
EC3.2.1.151 2001333
EC3.2.1.4
EGV EC3.2.1.4 GH45 Cel45A endoglucanase V inverting Saloheimo et CMC, PASC, Avicel, Glc3/Glc4/Glc5, barley β-glucan, glucomannan, filter paper Karlsson et al., 2002334
al., 1994335
Egl6 EC3.2.1.151 GH74 Cel74A endoglucanase and inverting Foreman et xyloglucan, hydroxyethylcellulose Benkő et al., 2008336
xyloglucanase al., 2003319
EG7 AA9 (formerly LPMO oxidative Foreman et cellulose Karkehabadi et al., 2008337
GH61) al., 2003319
Cel61B
Review

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currently in CAZy. Some families, for example GHs 6 and 7,
contain enzymes with vastly different and mechanistically
synergistic activities (as described in sections 6 and 7). Other
families, GHs 1 and 3, contain many enzymes with essentially
the same activity (cleavage of the glycosidic bond in cellobiose);
upon closer inspection, however, some of these enzymes have
subtle differences in substrate specificity. Family GH7 is
somewhat enigmatic in that it contains both a CBH (Cel7A)
and an EG (Cel7B) based on the same protein fold (folded β
sheet sandwich). In this case, the EG homologue has substrate
tunnel associated peptide loops of shorter length than its CBH
counterpart (see section 6). We also note that the EGs found in
GH families 5, 7, and 12 act on cellulose to leave the terminal
hydroxyl in the retaining configuration. Family GH45 EGs leave
the terminal hydroxyl in the inverted configuration.
For more comprehensive reviews of the early literature on T.
reesei enzymology and strain development, we refer the readers
to several major reviews.32,316 The following sections primarily
focus on the developments in fungal cellulases following the
initial structural determinations of each enzyme family (and
CBMs) toward understanding structure−function relationships.
5. CARBOHYDRATE-BINDING MODULES AND
LINKERS
Biomass-degrading enzymes work at solid−liquid interfaces, and
the concentration of catalytic units at the surface is directly
related to the extent of substrate turnover. Thus, many enzymes
that work on polysaccharides are multimodular with catalytic
function accomplished by a single or multiple CDs coupled to a
binding function via one or more CBMs; these two domains are
connected together by linker peptides of varying length and
structure. To date, 69 distinct families of CBMs have been
discovered and characterized according to the CAZy database
(family 33 CBMs have been reclassified as oxidative enzymes, as
discussed below).151−153,156 As many biomass-degrading fungi
commonly employ family 1 CBMs for plant cell wall
degradation, we review the history and developments in
structure−function studies primarily of this particular family of
CBMs in this section. Moreover, CBMs are connected to CDs
via linkers, and thus, the roles of these domains are also
reviewed. We note that the study of CBMs is vast, and here we
primarily focus on family 1 CBMs. We discuss other CBMs
mainly when findings are relevant to our collective under-
standing of general CBM behavior. For more general
perspectives on CBMs, readers are referred to several reviews
from the past decade.156,338−342
5.1. Family 1 Carbohydrate-Binding Modules
That many GHs contain both binding and catalytic function was
first reported in a study by Van Tilbeurgh et al.343 Therein,
papain was used to proteolytically cleave the TrCel7A enzyme
into two primary fragments: a 56 kDa domain that lost catalytic
activity on cellulose and retained full activity on small molecule
substrates (the CD), and a 10 kDa domain identified as the
glycosylated C-terminal domain (the CBM-linker).343 The
authors went on to show that the binding affinity of the 56
kDa domain to crystalline cellulose is significantly reduced
without the C-terminal domain. On the basis of these results,
the authors proposed that TrCel7A is a multimodular protein
with a binding and catalytic function.343 Following this
important discovery, Tomme et al. further characterized the
proteolysis products of TrCel7A and TrCel6A from papain
cleavage.344 They similarly demonstrated that both enzymes are
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Review
cleaved into small, glycosylated domains at the C- and N-termini
of Cel7A and Cel6A, respectively, both of which are bound with
high affinity to crystalline cellulose. Both “core” domains
isolated from the two cellulases retained activity against small
molecule substrates. This study,344 along with similar work the
same year on two bacterial cellulases from Cellulomonas f imi,345
solidified the concept that cellulases can exhibit multimodular
structures with CBDs. Upon the discovery of additional
carbohydrate-binding ligands beyond cellulose, the term CBD
was replaced with the broader concept of CBMs.156
Kraulis et al. solved the first structure of a family 1 CBM using
NMR spectroscopy in 1989, which is shown in Figure 12.346
Figure 12. TrCel7A family 1 CBM structure solved by Kraulis et al.346
Several residues of interest are highlighted including Tyr5, Asn29,
Tyr31, Tyr32, Gln34 on the hydrophilic, flat face of the CBM and the
two disulfide bonds in the protein. The structure forms an irregular,
triple-stranded β-sheet core. (B) Sequence alignment of the TrCel7A,
TrCel7B, and TrCel6A CBMs. The figure was generated with ESPript
(http://espript.ibcp.fr).347
The 36-residue TrCel7A CBM was prepared via solid-state
peptide synthesis. The structure revealed an irregular, triple-
stranded, antiparallel β-sheet arrangement with an amphiphilic
character with a large, hydrophilic flat face exhibiting three
tyrosine residues and a large number of polar amino acids, and a
hydrophobic face on the “wedge” portion of the CBM. The
CBM sequence contains four cysteine residues comprising two
disulfide bonds, and all three possible combinations were
investigated to determine the most likely pairing. The disulfide
bonds are also shown in Figure 12. Perhaps the most cited
observation from this original work is the presence of three
conserved aromatic residues on the flat, hydrophilic face of the
CBM (Tyr5, Tyr31, and Tyr32 in the TrCel7A CBM). As
discussed below in detail, this flat face is implicated as the
binding face to crystalline cellulose.
The family 1 CBM structure was followed by the structures of
many more CBMs from various families. By 2004, a paradigm
had emerged for three types of CBMs, which were categorized
as type A, B, and C CBMs in a seminal review paper from
Boraston and co-workers in 2004.156 Type A CBMs, which
include family 1, are characterized by flat faces that bind to
crystalline cellulose or chitin. Type B CBMs exhibit extended
grooves or clefts for binding single sugar chains, such as those
found in hemicellulose, pectin, or amorphous regions of
polymers such as cellulose or chitin. Lastly, type C CBMs are
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characterized as those that bind mono-, di-, and trisaccharides.
In 2004, three functions were attributed to CBMs: substrate
targeting, enhancements to enzyme−substrate proximity, and
disruption of the substrate.156 Boraston and co-workers
published a recent revision of the A−B−C CBM paradigm
wherein type B CBMs are classified as those that bind in endo-
mode and type C CBMs bind in exo-mode.338 type A CBMs
remain classified as previously.
With the publication of the landmark structural study from
Kraulis et al.,346 this enabled a very large body of work to be
conducted to investigate the function of family 1 CBMs.348−364
With the knowledge of a multidomain CBH structure, Ståhlberg
and co-workers proposed an initial model for CBH action in
1991 using TrCel7A as a model enzyme, which formed much of
the basis for our collective model of CBH action.348 By
examining the binding capacity of the intact TrCel7A, the CD
alone, and the CBM-linker alone, it was proposed that the CBM
is responsible for targeting the intact enzyme to crystalline
regions, thereby increasing the concentration of the CD to the
cellulose surface. The CBM was also proposed to enable two-
dimensional diffusion of the CBH enzyme on the cellulose
surface to enable efficient catalytic performance.348 Two-
dimensional diffusion of a type A family 2 CBM from C. f imi
was later measured using fluorescence recovery after photo-
bleaching experiments, with an estimated diffusion rate of 2 ×
10−11 to 1.2 × 10−10 cm2/s. To our knowledge, similar
experiments have not yet been conducted for family 1 CBMs.
Reinikainen et al. expressed several variants of the full-length
TrCel7A in yeast, focusing on mutations on the flat face
(Y492A, Y492D, and Y492H; this is Tyr31 in the CBM-only
sequence numbering) and on the hydrophobic wedge face
(P477R).349 As will be discussed in more detail in the GH7
section below (section 6), this was one of the first studies to
note that heterologous expression of fungal cellulases leads to
lower activity than enzymes expressed natively, in this case by
more than a factor of 2 in activity against crystalline cellulose.
This observation was attributed to a higher extent of
glycosylation from yeast expression as observed by a large
heterogeneous population of enzymes on a gel. Within the
heterologously expressed enzymes, the authors observed that
wild-type Cel7A and the Y492H mutant exhibit roughly
equivalent activity, whereas Y492A and Y492D result in a
significant decrease in activity against crystalline cellulose
accompanied by a concomitant decrease in binding affinity, as
measured at low temperature. The P477R mutation also
resulted in similarly low activity and binding affinity as
Y492A; however, as the authors note, this could be due to
structural changes to the CBM given the removal of a proline
residue. This was not clearly resolved until a later study from
Reinikainen et al., wherein Y492A, Y492H, and P477R were all
produced in the native organism with the native cbh1 gene
knocked out.350 Interestingly, the P477R mutant bound
similarly to the wild-type Cel7A enzyme, contrary to the same
enzyme produced heterologously. Both the Tyr492 mutants
bound to a lower extent than the wild-type enzyme, by
approximately 60%. At a concentration of 1 M MgSO4, all
mutants performed similarly to the wild-type enzyme on
crystalline cellulose, which the authors state suggests that the
hydrophobic effect plays a significant role in CBM binding to
the substrate. Given the similarity of the P477R results to the
wild-type enzyme and the reduction in affinity and activity with
mutations to Tyr492, this led the authors to suggest that the flat,
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Review
aromatic-containing face of the CBM is likely the binding face to
crystalline cellulose.350
Around the same time, Linder and co-workers published a
series of comprehensive studies wherein the TrCel7A CBM was
investigated in great detail with 6 alanine mutant CBMs
produced via solid-state peptide synthesis and examined with
binding affinity measurements and 2-D NMR spectroscopy,
including Y5A, P16R, N29A, Y31A, Y32A, and Q34A (Table
3).352 The individual point mutants of Tyr5 and Tyr32 both
Table 3. Free Energy of Binding for TrCel7A CBM Mutants
Relative to Wild-Type352
CBMs compareda ΔΔG (kJ/mol)
Cel7A wild-type → Cel7A P16R 1.2
Cel7A wild-type → Cel7A N29A 2.4
Cel7A wild-type → Cel7A Q34A 4.9
Cel7A wild-type → Cel7A Y31A 7.3
a
Values for Y5A and Y32A, which lost their affinity to cellulose, were
not determined.
completely lost affinity to cellulose. P16R was the least-affected
mutant, similar to results from Reinikainen et al.349,350 wherein
this amino acid position had the least effect on Cel7A activity.
The 2-D NMR spectroscopy suggests that the Y5A mutation
affects the overall compactness of the structure, which was later
verified by Mattinen et al. by solving NMR structures of this
CBM.356 In all other cases, the 2-D NMR spectra suggest
minimal changes only to the CBM structures upon single-point
mutations, which was later verified structurally for the Y31A and
Y32A CBMs.356 Additional NMR spectroscopy of the same
CBMs was conducted with cellohexaose present in solution by
Mattinen et al.357 From this study, the authors suggested that
the flat putative binding face of the family 1 CBM aligns in
parallel with cellohexaose, thus potentially serving as a model for
how the CBM binds to crystalline cellulose.357
An examination of the differences between the CBMs from
TrCel7A and TrCel7B closely followed these initial family 1
CBM structure−function studies.351,358,359 When comparing the
CBM binding affinity alone, it was observed that the CBMs from
these two enzymes exhibit significantly different binding
affinities, with the Cel7B CBM possessing a greater affinity to
crystalline cellulose. Starting with the Cel7A CBM sequence, the
authors made multiple mutations, and found that the Y5W
mutation alone can explain much of the differences in binding
affinity between the two CBMs351 (Table 4). A subsequent
Table 4. Relative Free Energies of Binding for the CBMs of
TrCel7A, TrCel7A Y5W, and TrCel7B351
CBMs compared ΔΔG (kJ/mol)
Cel7A wild-type → Cel7A Y5W −1.1
Cel7A Y5W → Cel7B wild-type −1.4
Cel7A wild-type → Cel7B wild-type −2.4
study from Srisodsuk et al. compared the binding affinity of a
hybrid Cel7A CD and linker with the Cel7B CBM, which
demonstrated slightly higher activity on BMCC.358 This study
was closely followed by the solution structure of the Cel7B
CBM.359 The structure is quite similar to the TrCel7A structure
with the primary exceptions of an additional disulfide bond
between Cys2 and Cys18, and a tryptophan residue at the 7-
position in place of Tyr5. The alignment of aromatic residues on
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the flat, hydrophilic surface of the CBM is quite similar.
Srisodsuk et al. also noted that the activity of the Cel7B-CBM-
containing chimeric enzyme with the Cel7A CD exhibited
higher activity.358 In a similar vein, Takashima et al. later
conducted an exhaustive study of CBM binding affinity
correlated to GH7 CBH activity using the H. grisea GH7
CBH (Figure 13).365 Therein, they demonstrated that
Figure 13. Correlation between the relative affinity constant (x-axis)
and activity on Avicel (y-axis) of each CBM mutant in the study.
Reprinted with permission from ref 365. Copyright 2007 Elsevier.
tryptophan residues in the “outer” positions on the CBM
(corresponding to Tyr5 and Tyr31 in the TrCel7A CBM)
yielded the highest binding affinity measured for the H. grisea
Cel7A CBM, and this correlated to the highest measured activity
of the full-length enzyme. The authors also found a nearly linear
correlation between activity on Avicel and CBM binding affinity,
highlighting the key relationship between CBMs and cellulolytic
performance.365
Linder and Teeri also examined both the binding and
reversibility of the TrCel7A CBM on crystalline cellulose using a
sensitive tritium-labeling approach combined with dilution
experiments.353 Therein, they produced the CBM in E. coli as
a chimera with the TrCel6A CBM-TrCel7A linker-TrCel7A
CBM construct, described in detail in another study.354 The
resulting protein was cleaved to isolate the Cel7A CBM and
included 11 residues from the linker domain. The authors
demonstrated that the binding of the CBM was temperature
dependent, with higher binding at lower temperatures (the
authors tested 4, 22, and 30 °C) (Figure 14). More importantly,
they demonstrated that the CBM binding was completely
reversible, addressing a question in the literature regarding if
CBMs could desorb from cellulose. The authors conclude that
the rate of adsorption and desorption of CBMs and the binding
affinity to cellulose should be optimally balanced to maximize
cellulase activity and minimize nonproductive binding.353
Interestingly, however, a subsequent study from Carrard and
Linder demonstrated that the TrCel6A CBM could not be
desorbed from cellulose over a temperature range from 4 to 50
°C, even 8 days after dilution, in stark contrast to the similar
TrCel7A CBM.360 To ascertain the differences, the authors
examined two key differences between the two CBMs: namely,
Tyr5 in the Cel7A CBM is a tryptophan in the Cel6A CBM, and
the latter has an extra disulfide bond. The W7Y mutation in the
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Figure 14. T. reesei CBM binding temperature dependence: 3 h at 4 °C
(●), 18 h at 4 °C (○), 3 h at 22 °C (×), 18 h at 22 °C (■), 43 h at 22
°C (▲), 3 h at 50 °C (□), and 18 h at 50 °C (Δ). ▼ corresponds to
CBHI CBD, and ▽ to CBHII CBD, both at 18 h at 22 °C. Reprinted
with permission from ref 354. Copyright 1996 American Society for
Biochemistry and Molecular Biology.
Cel6A CBM led to a dramatic loss in binding affinity relative to
the wild-type Cel7A CBM. The removal of the third disulfide
bond in the Cel6A CBM resulted in a decrease in binding
affinity and off-rate, although not as drastic as the W7Y
mutation, suggesting that the Cel6A CBM rigidity contributes to
the CBM-cellulose interaction and that the presence of the
tryptophan at the 7-position significantly affects the binding
affinity.360 In a separate study, the same authors demonstrated
that the native TrCel7A CBM binding affinity was not
significantly affected by pH.361
More recently, Guo and Catchmark conducted a thorough
study using isothermal titration calorimetry and adsorption
isotherms to compare the binding characteristics of TrCel7A
and TrCel6A CBMs expressed in E. coli on crystalline cellulose,
Avicel, PASC, and small cellodextrin chains.364 To enable
quantitative comparison of binding affinity across various
cellulose substrates, the authors first developed a methodology
to quantify the available surface area for CBM binding based on
nitrogen adsorption and static light scattering. Similar to
previous conclusions from Carrard and Linder,360 the authors
demonstrated that the binding affinity of the TrCel6A CBM is
significantly higher, specifically by an order of magnitude relative
to the TrCel7A CBM (106 M−1 versus 105 M−1, respectively).364
The application of isothermal titration calorimetry enabled the
delineation between enthalpic and entropic contributions to
binding affinity for the first time for family 1 CBMs, which
demonstrated that the binding affinity to the cellulose surface
was enthalpically favorable and entropically unfavorable. This
finding is in direct contrast to an influential, earlier study
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wherein isothermal titration calorimetry experiments on a family
2 CBM (also a type A CBM) suggested that type A CBM
binding is entropically driven.366 Interestingly, Guo and
Catchmark also examine the binding specificity of both CBMs
to NaBH4-treated cellulose microfibrils, which modify cellulose
chain reducing ends, finding that the Cel6A CBM exhibits much
lower binding affinity.364 They attribute this difference to the
fact that the Cel6A CBM may recognize the reducing ends of
cellulose chains, whereas significantly less difference in binding
was observed for the Cel7A CBM. The authors speculate that
the potential preference in the Cel6A CBM binding may lead
the intact enzyme to bind near reducing ends of cellulose
chains.364
In 2003, the binding-face specificity of two families of type A
CBMs, including a large library of family 1 CBMs and a family 3
CBM from C. thermocellum, to cellulose was reported.362 Lehtiö
et al. fused CBMs of interest to a modified staphylococcal
protein A, which was then coupled to an immuno-gold label. By
binding the CBMs to very large cellulose Iα microfibrils from
Valonia, the authors could then visualize the specific cellulose
binding face for the CBMs directly with TEM and diffraction
measurements. It was shown that in all cases the CBMs bound
to the 110 face of cellulose Iα (Figure 15), which is the
Figure 15. Crystal structure faces of the cellulose Iα polymorph. The
circle indicates the 110 face, exposed in worn crystals, which is the
CBM binding site. Reprinted with permission from ref 362. Copyright
2003 National Academy of Sciences.
hydrophobic face (the hydrophobic face in cellulose Iβ is 100).
This finding shed light directly on the type A CBM-cellulose
interaction by ascertaining that the protein−ligand binding
surface is the flat surface with the glucopyranose rings directly
exposed. It should be noted that this type of face only exists in
the natural cellulose I polymorphs with multiple chains
exhibiting exposed faces; cellulose II and III do not exhibit
“flat” faces with multiple parallel chain faces exposed.88−90,93,94
The authors also measured the binding affinity of multiple
CBMs and noted that the addition of two tryptophan residues to
the TrCel7A CBM does not increase the binding affinity beyond
the effect of a single Y-to-W mutation.362 Unsurprisingly, later
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Review
imaging work with intact TrCel7A also suggested that the whole
enzyme binds to and attacks cellulose on the hydrophobic
surface, likely mediated by the CBM.367
More recently, Sugimoto et al. examined the family 1 CBM
binding behavior to cellulose in more quantitative detail.363 The
authors fused a fluorescent protein with the TrCel7A CBM and
measured binding isotherms to various crystalline and
amorphous cellulose substrates. The authors fit their binding
isotherms to four binding models, and found that the Hill
binding model provided the best fit for CBM-binding to
cellulose, with the Langmuir isotherm model also providing a
reasonable fit (correlation coefficients of 0.9995 and 0.9915,
respectively) at 5 °C on Cladophora cellulose. The authors
attribute the goodness-of-fit of the Hill model to the explicit
treatment of a steric exclusion effect induced by surface
crowding of the CBM-fluorescent protein complex and infer
that the length and flexibility of the linker domain will dictate
the size of the exclusion area.363
To date, nearly all studies of family 1 CBMs have either used
solid-state peptide synthesis or E. coli expression, neither of
which impart glycosylation to the protein. However, detailed
mass spectrometry studies from Harrison et al. in 1998 showed
that the TrCel7A family 1 CBM exhibits glycosylation at the
Thr1 and Ser3 positions, as illustrated in Figure 16.368 Mass
Figure 16. (A) Glycosylated TrCel7A CBM on the hydrophobic
surface of cellulose, as studied by Taylor et al.359 (B) LOGO
representation372 of a multiple sequence alignment of family 1 CBMs
suggests that multiple putative O-glycosylation sites may exist on family
1 CBMs.
spectrometry analysis was not conducted beyond Tyr5. Given
the importance of glycosylation in cellulase activity,369 as
discussed in more detail below in several GH sections, and the
sequence conservation of putative O-glycan sites on family 1
CBMs (Figure 16),370 fungi may employ glycans on CBMs for
multiple functions. In terms of cellulose binding affinity, Taylor
et al. employed thermodynamic cycle calculations with
molecular dynamics (MD) simulations to examine how O-
glycans, especially at Ser3 and a conserved site at Ser14, are able
to modify the binding affinity over the nonglycosylated
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variant.371 The predictive capability of these simulations was
first tested by comparing to previous experimental data for
amino acid substitutions. The thermodynamic cycle calculations
were in quantitative agreement, for example, with the Y5W
mutation. From there, the authors predicted that a single
mannose at Ser3 could modify the binding affinity by the same
order as a tryptophan mutation. Moreover, the simulations
predicted that the addition of mannosylation at different sites
would modulate the binding affinity by a significantly different
magnitude, suggesting that the location and extent of
glycosylation has a major impact on the change in properties.371
The predictions that glycosylation affects CBM binding
affinities were recently tested experimentally.373 Chen et al.
developed a solid-state glycopeptide synthesis approach to
rapidly produce single glycoforms of the TrCel7A CBM. Two
sets of glycoforms (for a total of 20 CBMs) were synthesized:
the first set focused on addition of glycan motifs to single amino
acids, namely at Thr1, Ser3, and Ser14 of mono-, di-, and
trimannose group, whereas the second set was designed to study
the effects of adding glycans to multiple amino acid residues
simultaneously, as would be found naturally.368 For each
glycoform, the thermal stability, resistance to thermolysin
cleavage, and the binding affinity to BMCC were tested. For
the addition of glycans to single sites, Chen et al. discovered that
Ser3 has the largest proteolysis-stabilizing effect (by up to a
factor of 10), increase in thermal stability (up to 12 °C), and
binding affinity increase to BMCC (up to a factor of 4).373
Addition of glycans at Thr1 does not demonstrate significant
differences in proteolytic and thermal stability, but a
disaccharide at Thr1 is able to increase the binding affinity
significantly to BMCC. Addition of glycans at Ser14 essentially
improved all properties, but not to the extent of Ser3. For the
addition of glycans to multiple sites, single mannosylation at
each site was able to increase the binding affinity to BMCC by
7.4-fold, well over the increases demonstrated via amino acid
substitutions.351,352 An increase of up to 50-fold in thermolysin
resistance was demonstrated with a concomitant increase in
thermal stability of up to 16 °C for several variants. From a
biological perspective, glycan-bearing residues are highly
conserved in the 1, 3, and 14 positions on family 1 CBMs,
and given the ability to affect multiple, beneficial properties, it is
likely that CBM glycosylation is employed commonly by fungi.
Given the prevalence of glycosylation in fungal cellulases, this
work demonstrates that the study of CBMs should explicitly
consider the effects of glycosylation to measure physiologically
relevant properties.373
Another interesting question related to CBMs is the ability for
substrate disruption, as discussed by Boraston et al.156 Din et al.
published an early study on CBM substrate disruption with the
family 2 CBM from the C. f imi EG, CenA.374 Therein, they
observed that application of the CBM-linker domain on ramie
cotton fibers resulted in surface roughening. The authors
suggested that this CBM-cellulose interaction was the result of
nonhydrolytic disruption of the substrate. However, it was not
demonstrated that the CBM-treated substrates were subse-
quently more digestible by reducing-sugar assays nor were
cellulose crystallinity measurements conducted. Ståhlberg et al.
incubated the TrCel7A CBM-linker domain with Avicel, but did
not observe additional susceptibility of the resulting substrate to
digestion by intact Cel7A.348 Several subsequent studies have
reported observing substrate disruption. Gao et al. claimed that
the Penicillium janthinellum CBM-linker was able to synergize
with an EG from T. pseudokoningii on Avicel and cotton fibers,
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but no data were included in their study to quantitatively
support this claim.375 Instead, the authors show scanning
electron microscopy (SEM) images of before and after
treatment with the P. janthinellum CBM-linker domain. Xiao
et al.376 and Wang et al.377 report similar findings, also without
demonstrating synergistic cellulose depolymerization for the
CBM from a T. reesei EG and from a family 7 CBH from T.
pseudokoningii, respectively. These studies overall lack sufficient
detail to substantiate the interpretations of SEM and FTIR data.
More recently, Hall and co-workers reported an in-depth study
of “CBM pretreatment” of Avicel and fibrous cellulose (cotton
linters).378 Therein, they noted that a 15 h pretreatment at 42
°C followed by enzymatic hydrolysis at 50 °C was able to
slightly improve the digestibility of Avicel and was able to
slightly decrease the crystallinity as measured by the height of
the 200 peak in X-ray diffraction measurements. It is unknown
how pretreatment with CBMs affected the conversion at long
times or the final yield of reducing sugars from these
experiments. Despite continued claims of substrate disrup-
tion,379 the data for a disruption effect remain somewhat sparse,
and a full study of CBM disruption in terms of synergy with
cellulases at high substrate conversions coupled to detailed
substrate characterization does not yet exist, thus severely
limiting the ability to ascertain true “disruption” effects by
CBMs.
Many other CBM effects on fungal cellulase activity and
behavior beyond substrate disruption have been investigated.
Hall and co-workers compared the thermal stability of intact
TrCel7A, the CD alone, and the CBM-linker, the latter two
isolated from papain cleavage of the intact enzyme.380 They
found melting temperatures of 59, 51, and 66 °C, respectively,
suggesting that the CBM-linker is a significant stabilizer of the
intact enzyme. Voutilainen et al. produced chimeric enzymes of
the Thermoascus aurantiacus Cel7A (TaCel7A) CBH, which
natively lacks a CBM and linker, to either the TrCel7A or
Chaetomium thermophilum Cel7A CBM-linker.381 On Avicel at
high temperature, this resulted in a significant increase in
activity, suggesting that CBM-linker pairs from thermostable
cellulases confer disparate benefits in terms of thermal stability
to chimeric CBHs.
Voutilainen et al. also published a recent study wherein they
built chimeras with CBMs from Families 1, 2, and 3 of the
thermostable Talaromyces emersonii Cel7A (TeCel7A) enzyme
and an engineered TeCel7A, both of which natively lack a
CBM.290 This study revealed that the CBM3-containing
enzymes bind approximately 20% more to Avicel over the
family 1 and family 2 CBM-containing enzymes at both 45 and
60 °C. Correspondingly, the family 3 CBM-containing cellulases
are able to solubilize a greater amount of Avicel in 24 h across a
range of temperatures from 45 to 65 °C. A similar study was
reported from Kim et al. wherein a large library of EGs were
expressed with varying CBMs.382 The authors found that the
activity of the EG increases generally with the addition of CBMs,
and that the thermal stability in some cases, even with the
addition of CBMs and linkers from mesophilic organisms, was
improved.
Although many cellulases have CBMs, there are many
examples of fungal and nonfungal cellulases that do not employ
them in natural biomass degradation. There are likely multiple
physiological and evolutionary reasons for the lack of CBMs in
some cases, such as biomass degradation in organisms that
densely pack solids into their digestive organs.383 Recently, an
elegant study from Várnai et al. shed light on one potential
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reason by examining the performance of a T. reesei enzyme
cocktail at high solids loadings.384 Therein, they demonstrated
that T. reesei cellulases with their CBMs and linkers removed are
able to achieve the same extents of conversion on both Avicel
and pretreated wheat straw at 20% solids loading. The most
likely reason for this observation is that, at high solids loadings,
the diffusion length for the enzymes to productively bind to
substrate is much shorter than in low solids loading digestions,
thus precluding the need for CBMs to ensure that the
concentration of active enzymes at the substrate interface is
high. For natural systems, high dry-matter content may also
preclude the need for CBMs, but a systematic correlation
coupled to other environmental and evolutionary considerations
has not been conducted to our knowledge. For commercial
biomass conversion, the work from Várnai et al. suggests that
additional research should be conducted to understand the need
or lack thereof for CBMs in cellulolytic enzymes in industrial
contexts, as this could lower the overall mass loading of enzymes
for biomass degradation to sugars.384
Given the relatively small size of family 1 CBMs coupled to
the inherent difficulty in directly studying molecular-level
interactions at the heterogeneous cellulose interface, family 1
CBMs have been the subject of computational studies since
1995 aimed at further elucidating cellulose−CBM interactions
at the molecular level.121,123,370,371,385−395 Nimlos et al.
conducted MD simulations of the TrCel7A CBM on the
hydrophobic face of cellulose and observed that Tyr13
undergoes a conformational change from being tucked into
the CBM structure to bind directly to the substrate. It is
noteworthy that this simulation was conducted with a previous
version of the CHARMM force field,396 and this behavior has
not been observed in subsequent simulations with more updated
potentials. The conservation of an aromatic residue in this
position (Figure 16), however, suggests that this aromatic
residue is important for a structural or functional reason, and
future experimental work will likely shed additional light on this
question. Multiple studies were later reported that suggest how
CBMs translate on the cellulose surface.121,123,370,392,394,395
Potential energy surfaces of a family 1 CBM on both a coarse-
grained and atomistic surface of cellulose Iβ revealed that the
Cel7A CBM displays potential energy wells every cellobiose unit
(roughly 1 nm) over the surface of cellulose (Figure 17). A
much more detailed study was later published by Nimlos et al.
describing multiple aspects of CBM behavior on cellulose. This
study demonstrated that there is a thermodynamic driving force
for the TrCel7A CBM to translate from hydrophilic surfaces to
Figure 17. Simulation of a family 1 CBM of a cellulose Iβ surface was
used to calculate potential energy surfaces as shown. The minimum
energy wells, shown in dark blue and violet, represent preferential
locations for the CBM at the surface. These locations correspond to
approximately 1 nm, which is the length of a cellobiose unit. Reprinted
with permission from ref 370. Copyright 2010 American Chemical
Society.
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the preferred hydrophobic face, that the CBM will translate
along the hydrophobic surface in both a forward and backward
direction with equal probability, and that the flat, hydrophilic
face of the CBM is the thermodynamically preferred face for
cellulose binding.
Taken together, significant insights have been gained
regarding the function of family 1 CBMs, especially accelerated
and informed by the initial structural work.346 Yet, these studies
also illustrate that our collective understanding of CBM function
for activity and stability in the context of full-length, multi-
modular enzymes and the corresponding potential for protein
engineering via CBMs remain limited. Moreover, as is clear from
this section as well, most of our collective understanding of
family 1 CBMs stems almost solely from the model T. reesei
system, despite the prevalence of family 1 CBMs in many other
fungi. Undoubtedly, there is significant potential for improving
cellulase properties via deeper understanding of CBM function
in the context of both cellulase performance and stabilization.
5.2. Linkers
In multimodular cellulases, linkers of various length and
sequence diversity connect CBMs to CDs. In some cases,
these linkers are very short, and the CBMs are thus in intimate
contact with the CDs.397,398 In fungi, however, linkers
characterized to date tend to be longer and exhibit glycosylation,
thus allowing greater separation between CBMs and CDs. Here,
we review the characterization of linkers and their putative
functions beyond domain connection. We include discussion of
nonfungal linkers where appropriate to understanding their
general function.
Some of the original work related to linker domains and
cellulase modularity was conducted with small-angle X-ray
scattering (SAXS) using T. reesei and C. f imi cellulases as model
systems.399−403 Abuja and co-workers examined both Cel7A and
Cel6A, and in both cases, determined that the enzymes
exhibited a “tadpole”-like structure with a large core domain
(the CD) and a long, flexible “tail” (the CBM-linker; Figure
18).399 On the basis of the work from van Tilbeurgh et al.,343
there was already strong experimental evidence indicating that
the “tail” domain was responsible for carbohydrate binding. A
“collar”-like section in the Cel7A linker region was also
Figure 18. Overall “tadpole” topologies of the TrCel7A and TrCel6A
enzymes. The C- and N-termini are noted, highlighting the reverse
locations of their cores (CDs). The Cel7A linker displays a
characteristic “girdle”, while the Cel6A linker comprises two repeating
units. Adapted with permission from ref 399. Copyright 1998 Elsevier.
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attributed to the presence of O-glycosylation.400 It was noted
that the TrCel6A tail region was significantly longer than its
GH7 counterpart from the same organism. A second study on
TrCel7A demonstrated that the enzyme becomes elongated in
the presence of xylan from 18 to 22 nm, which was attributed to
lengthening in the CBM-linker region.401
Langsford et al. published the seminal study demonstrating
glycosylation of cellulase linkers in C. fimi serves to protect
against proteolysis.404 Specifically, the authors isolated two
cellulases from C. f imi and expressed their counterparts in E. coli,
the latter of which does not natively glycosylate proteins. The
properties of the enzymes in terms of kinetics on small molecule
substrates (such as CMC or pNPC) and thermal and pH
stabilities were not affected by glycosylation.404 Binding was
measured at 0 °C on Avicel and also suggested that, at those
conditions, glycosylation had no effect. In the presence of crude
protease from C. f imi, however, the recombinant cellulases
showed fragmentation patterns that suggested the proline-
threonine rich linker domains are proteolytically cleaved.
Cellulase performance in Avicel digestion was not directly
compared in the absence of protease. Overall, this study led
Langsford et al. to suggest that the C. f imi cellulases were
multimodular with CBMs and CDs, similar to that of van
Tilbeurgh et al.343 Importantly, they also suggested that the
proline-threonine rich domain between these two functional
domains is a “hinge region” (now commonly referred to as the
“linker”) that contains O-glycosylation that protects against
protease action.404 Although an equivalent study has not been
conducted to our knowledge in fungi, perhaps due to the
inherent difficulties in expressing fungal cellulases in E. coli or
fully deglycosylating fungal cellulases, it is likely that the finding
from Langsford and co-workers is similar in multimodular
cellulases regardless of origin in that O-glycosylation on linker
domains tends to shield the linkers from proteolysis during
cellulose depolymerization in an extracellular, competitive
environment.
Srisodsuk et al. examined the activity of TrCel7A as a function
of linker length.251 Therein, the authors produced two mutant
enzymes, one in which the approximately first one-third of the
linker was removed, comprising Gly434-Gly444, referred to by
the authors as the “hinge” region, and the other from Gly434 to
Gly 460, which essentially is removal of the entire linker. The
hinge mutation resulted in similar overall performance of the
enzyme relative to the wild-type, but with a reduced binding
capacity at high loading. The mutant removing the entire linker
drastically reduced the enzymatic activity toward crystalline
cellulose. The overall interpretation of the linker’s role by the
authors was that it is important for maintaining sufficient
distance between the CBM and CD and that it facilitates
“dynamic adsorption” to the surface of cellulose.251 An earlier,
similar study on a bacterial cellulase also demonstrated that
removal of the linker domain significantly reduces its activity
toward both crystalline and amorphous cellulose.405
Boisset et al. examined the H. insolens EG V (GH45 cellulase)
with static light scattering, which contains a family 1 CBM and a
33-residue linker.406 They found that the average length of each
residue in the linker domain is approximately 2 Å, which is not
in agreement with α-helices, polyproline helices, or β-sheets.
From these initial SAXS and light scattering studies, it was not
clear if linkers are stiff or flexible. Receveur and co-workers later
reported a comprehensive SAXS study wherein they examined
the full-length H. insolens GH45 EG (the same enzyme as from
Boisset et al.406), the CD alone, the enzyme with the CBM
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removed, the CBM alone, a variant where a substantial portion
of the linker was removed, and a polyproline insertion in the
linker.407 They found that the CBM presence does not alter the
overall conformation of the enzyme and that the CBM was not
discernible from the linker region. The results for the wild-type
enzyme and the shortened-linker variant both demonstrate that
the linker volume is quite substantial and extended, suggesting
either (or both) that the glycosylation on the linker provides an
excluded volume effect or that the linker is quite flexible, but
extended. The polyproline mutant exhibited a considerable
narrowing in the region where the proline insertion was made,
suggesting that this region imparted significant local rigidity.
The authors interpret their data to suggest that the linker
provides the means to separate the CBM and CD and optimize
their relative geometry, similar to the interpretation of Srisodsuk
et al.251 The authors go on to state that the conformational
landscape of the linker may impart a “caterpillar”-like motion
between the CBM and CD during catalytic action on insoluble
cellulose. This model is one wherein free energy is gained by
compression of the linker during catalysis, which is dissipated by
sliding of the CBM.407
In the aforementioned study from Receveur et al.,407 the
authors could not resolve the CBM in the full-length H. insolens
GH45 EG, thus limiting their ability to decipher linker behavior
relative to CBM behavior in their SAXS experiments. To
overcome this problem and focus only on the linker domain,
von Ossowski et al. built a chimeric cellulase with 2 GH6
cellulases from H. insolens (Cel6A and Cel6B) with an 88-
residue linker between them, wherein both the CDs were
completely discernible from the linker region.408 Using SAXS
combined with molecular modeling, the authors demonstrated
that the chimeric cellulase adopts a huge range of conformations
in solution accessible at low energetic cost with equal probability
(thus, free energetically similar) from compressed to extended,
as measured by the end-to-end distance. To our knowledge, this
study marked the first explicit connection of linkers with
intrinsically disordered proteins, which has become a large field
in the last 10−15 years.409−413 The authors also propose that the
O-glycans may provide greater extension between the
subdomains, and that their results provide additional evidence
toward an inchworm- or caterpillar-like mechanism. Additional
studies have been published that include SAXS analysis of intact
cellulases with broadly similar conclusions.414
Besides SAXS, NMR and MD simulation also been used to
characterize linker behavior. Poon et al. used NMR spectroscopy
to examine the proline-threonine rich linker of a family 10
xylanase from C. f imi.415 Therein, they showed that the linker
samples conformational space on very fast time scales: from
picoseconds to nanoseconds, and that glycans serve to dampen
mobility. In two subsequent papers, we examined linker
behavior in 3 GH7 cellulases and the TrCel6A linker.416,417
Beckham et al. first used replica-exchange MD to examine the
TrCel7A linker with and without O-glycosylation.416 It was
shown that the Cel7A linker was an intrinsically disordered
protein in solution and that the primary role of the glycans in the
isolated domain was to serve an excluded volume effect, as
measured by the conformational free energy of the linkers as a
function of the end-to-end distance. A later study from
Sammond et al. demonstrated similar behavior for three
additional fungal cellulase linkers.417 Therein, the lack of
structure predicted by replica-exchange MD simulation was
confirmed for the nonglycosylated variants of all four linkers
with circular dichroism spectroscopy.
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Figure 19. Characteristics of cellulase linkers; data adapted from Sammond et al.417 (A) Linker length histograms for fungal GH7 and GH6 cellulases.
(B) O-glycosylation distribution across linker sequences measured by the prevalence of serine and threonine residues for fungal GH7 and GH6
cellulases.
It has often been mentioned that linkers in cellulase enzymes
do not exhibit sequence conservation or high homology to one
another. In the aforementioned study from Sammond et al., an
alternative set of methods was applied to study the similarities
and differences between linkers besides sequence alignments.417
Instead of the conventional sequence identity, linker lengths,
amino acid content, glycosylation distributions, and these
variables as a function of one another, of the GH family, and
of the origin (i.e., either bacterial or fungal) were compared. In
doing so, quantitative patterns in linker characteristics begin to
emerge, some of which are illustrated in Figure 19.417 For
example, multimodular fungal GH6 and GH7 cellulases exhibit a
significant difference in linker length (with GH6 cellulase linker
average length of 42 and GH7s at 30 residues), the functional
reason for which remains unknown. In all cases examined, O-
glycosylation was found to be approximately uniformly
distributed across the length of linkers, suggesting that it is
needed in a distributed manner to protect against proteolysis or
to serve additional, unknown functions. Glycine residues were
found to be clustered at the termini of all linkers studied,
suggesting the need for flexibility at the junction between
ordered domains and the linker, perhaps for orientation during
catalytic action. The amino acid content of bacterial and fungal
linkers differed significantly, with higher proline content in
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bacterial cellulase linkers and higher Ser/Thr content in fungal
cellulase linkers. Taken together, these results begin to suggest
that cellulase linkers exhibit properties that are optimized for
specific catalytic function, the mechanistic underpinnings for
which are mostly unknown.417 Given the wealth of sequence
data for cellulases and other carbohydrate-active enzymes in the
CAZy database,151−153 this bioinformatics approach offers a
straightforward, quantitative means to develop new hypotheses
regarding linker function and should provide a framework for
the comparison of linkers beyond simple sequence alignments.
Detailed characterization of the glycosylation pattern of
linkers is essential to understand their behavior. To that end,
several in-depth mass spectrometry studies have been conducted
to characterize the glycosylation patterns on cellulase linkers.
For TrCel7A, Harrison et al. published the first detailed study of
the linker domain glycosylation in 1998; the primary results
from which are shown in Figure 20.368 Therein, the authors
examined Cel7A from a hyper-cellulase-producing strain of T.
reesei (ALKO2877) and determined that all serine and threonine
residues in the linker exhibited at least a single O-mannose
residue. They also found that a mannose residue on the linker
exhibited a sulfate group, but the role of this sulfation remains
unknown. In 2001, Hui et al. characterized the linker of the same
enzyme produced in RUT-C30, Iogen-M4, and Iogen-B13 T.
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Figure 20. (A) TrCel7A linker glycosylation pattern from Harrison et
al.368 (B) Molecular snapshot of the linker from Beckham et al.416
reesei strains.273 In all cases, the serine and threonine residues
exhibited heterogeneous mannose distributions, with a single
phosphorylation on an undetermined site in the linker. Shortly
after, Hui et al. published a second study characterizing the
glycosylation of TrCel6A, TrCel7B, and of the GH5 EG,
TrCel5A.418 For each linker, the following number of glycans
were detected on each linker: 39−46 O-glycans for Cel6A, 24−
34 for Cel7B, and 32−42 for Cel5A. Stals et al. conducted a
thorough examination of glycosylation on the TrCel7A linker as
a function of growth conditions and found that, in minimal
media at low pH, 19−29 mannose residues were present,
whereas, in rich media, the O-glycans on the linker were
trimmed back to 16−23 residues.419 On the basis of analysis of
fungal glycosylation pathways and the aforementioned mass
spectrometry data, the T. reesei O-glycans are likely primarily
mannose units connected by α-O linkages to serine or
threonine.273,368,418−422 However, other carbohydrate mono-
mers, such as glucose, as well as branching and straight chain O-
glycans are known to be found in yeast and fungi, including T.
reesei.423 Mannose and other carbohydrate monomers are
known also to be connected by both α2 and α6 linkages.
These data, when taken together, further complicate the ability
to examine the impact of linker glycosylation in a systematic
manner.
Many studies have been conducted to understand how linkers
behave in isolation or how intact multimodular enzymes behave
in solution. Conversely, our collective understanding of linker
function during enzymatic action is limited given that cellulases
act at a solid−liquid interface, which is inherently difficult to
study at the molecular level. Related to cellulase action vis-à-vis
linker function, Receveur et al. proposed the inchworm
hypothesis wherein, during catalytic action, the linker will
Figure 21. Molecular snapshots of TrCel7A and TrCel6A wherein the linker binds to the cellulose surface from microsecond-long MD simulations.
These computational predictions of cellulose linkers enhancing binding of CBMs to the cellulose surface were corroborated experimentally via binding
isotherm measurements.393
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store free energy that is dissipated during catalysis or CBM
movement, thus driving the enzyme forward. This hypothesis
was tested computationally by using a free energy method
(umbrella sampling) by compressing and extending an isolated
linker domain over a cellulose slab, which suggested that there
was indeed a barrier.424 However, the computed barrier for
compression was found to be incredibly high, likely given that
convergence of simulations of these types are quite difficult with
such a low-resolution reaction coordinate as end-to-end distance
of a large, glycosylated linker peptide.416,424
Ting et al. proposed a theoretical mechanochemical model of
how a multimodular CBH such as TrCel7A can work at the
solid−liquid interface.425 Therein, they assumed that the CBM
and CD are random walkers connected by a linker modeled as a
spring. The possible steps in the model, governed by a master
equation, are CBM motion in a “forward” or “backward”
direction, and CD motion (once productively bound to a
cellulose chain) in a “forward” direction, i.e., a hydrolysis and
processivity event. Note that the events of processive cellulolytic
action are described in detail in sections 6.2 and 7.2.7. The rate
constant governing hydrolysis and processivity for the CD
account for both the work required to decrystallize a single
polymer chain and the compression of the linker as the CD
moves forward. The model demonstrates that the maximum
enzyme velocity on the surface is reached at intermediate linker
stiffness, for a given length. The overall recommendations from
this theoretical model are that optimization needs to be
conducted not only of the CD hydrolysis rates, as was already
known, but also of the linker length and stiffness.
Toward further understanding linker behavior during
cellulase action, we recently reported a combined computational
and experimental study wherein the interaction of the TrCel7A
and TrCel6A enzymes with the surface of cellulose were
examined.393 From long MD simulations, it was predicted that
the glycosylated linkers in both enzymes were able to bind to
cellulose, as illustrated in Figure 21. Subsequently, the binding
affinities to cellulose were experimentally measured of both the
glycosylated TrCel7A CBM-linker (isolated via papain cleavage)
and the CBM alone (produced with solid-state peptide
synthesis). These experimental measurements demonstrate
that the linker indeed is able to increase the binding affinity to
cellulose by a factor of 10 over the CBM alone, as fits with a
Langmuir isotherm model, and that it likely does not function as
a spring between the two structured domains. The MD
simulations further revealed that the linker binding is dynamic,
and the linker lacks secondary structure upon binding, thus
suggesting that its binding mechanism is one that is nonspecific
compared to that of the CBM. These results hearken back to the
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 Table 5. Summary of Biochemical Characterizations of Fungal GH7 Cellulases
temp
opt (°
organism expression host enzyme pH opt C) substrate for opt substrate specificity ref comments
Acremonium ther- Trichoderma ree- Cel7A 5.0 60 4-methylumbelliferyl- 4-methylumbelliferyl-β-D-lactoside, 2-chloro-4-nitrophenol- Voutilainen et al., 2008,381
mophilum sei β-D-lactoside β-D-lactoside, Avicel, PASC, filter paper, hydroxyethylcel- Szijártó et al., 2011483
ALKO4245 lulose, xylan, p-nitrophenyl-β-D-glucoside
Chemical Reviews

Acremonium sp. Cel7A pNPC, pNPL, PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., 2010332 assay condition 50 °C and pH 5.0;
CBS265.95 CMC, xyloglucan, xylan, arabinoxylan, mannan, galacto- 72% residual activity on CMC after
mannan 3 h at 40 °C and pH 5.0
Acremonium sp. Cel7B pNPC, pNPL, PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., 2010332 assay conditions 50 °C and pH 5.0;
CBS265.95 CMC, xyloglucan, xylan, arabinoxylan, mannan, galacto- 92% residual activity on CMC after
mannan 3 h at 40 °C and pH 5.0
Aspergillus aculeatus CBHI 3.0 60 Avicel Avicel, insoluble oligosaccharides (DP20), CMC, alkali- Takada et al., 1998,484,485 Ka- identical optima, but lower activity,
F-50 swollen cellulose, pNPL namasa et al., 2003486 when expressed in S. cervisiae
Aspergillus nidulans Pichia pastoris CBHI, CMC, Glc3/Glc4/Glc5/Glc6 Bauer et al., 2006487 assay conditions 37 °C and pH 4.5
FGSC A4 CbhB
Aspergillus nidulans Pichia pastoris EglB 5.5 42 CMC CMC, Glc4/Glc5/Glc6, barley β-glucan, lichenan, xyloglucan Bauer et al., 2006487
FGSC A4
Aspergillus niger CbhA CMC Gielkens et al., 1999488 assay conditions 30 °C, using parti-
CBS 513.88 ally purified enzyme
488
Aspergillus niger CbhB CMC Gielkens et al., 1999 assay conditions 30 °C, using parti-
CBS 513.88 ally purified enzyme
Aspergillus oryzae CelB 4.0 45 CMC CMC, barley β-glucan Kitamoto et al., 1996,489 Kotaka stable below 50 °C and at pH
et al., 2008490 3.0−7.0; A. oryzae KBN616 used
for CMC activity; A. oryzae RIB40
used for barley β-glucan activity

1335
Bispora sp. MEY-1 Pichia pastoris Bgl7A 5.0 60 lichenan barley β-glucan, lichenan, CMC, laminarin, xylan Luo et al., 2010,491 Zhang et al. stable at 60 °C for at least 1 h and at
2013492 pH 1.0−8.0
Chaetomium ther- Trichoderma ree- Cel7A 4.0 65 4-methylumbelliferyl- 4-methylumbelliferyl-β-D-lactoside, 2-chloro-4-nitrophenol- Voutilainen et al., 2008,381
mophilum sei β-D-lactoside β-D-lactoside, Avicel, PASC, filter paper, hydroxyethylcel- Szijártó et al., 2011483
lulose, xylan, p-nitrophenyl-β-D-glucoside
Chaetomium ther- Pichia pastoris Cbh3 5.0 60 pNPC pNPC, MCC, filter paper Li et al., 2009493
mophilum CT2
Chrysosporium luck- Cel7A 5.0−5.5 pNPL pNPL, <italic>p</italic>NPC, Avicel, CMC, cotton Gusakov et al., 2005494 assay conditions 40 °C; maintained
nowense >90% activity after 7 h at 60 °C
Cladorrhinum foe- Cel7 pNPC, pNPL, PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., 2010332 assay conditions 50 °C and pH 5.0;
cundissimum CMC, xyloglucan, xylan, arabinoxylan, mannan, galacto- 30% residual activity on CMC after
mannan 3 h at 60 °C and pH 5.0
Claviceps purpurea Cel1 CMC, Avicel Müller et al., 2007495
T5
Fusarium oxysporum EG I, pNPC, pNPL, PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., 2010332 assay conditions 50 °C and pH 5.0;
Cel7B CMC, xyloglucan, xylan, arabinoxylan, mannan, galacto- no detectable residual activity on
mannan CMC after 3 h at 60 °C and pH 5.0
Fusicoccum sp. Pichia pastoris CBHI 5.0 40 4-methylumbelliferyl- Avicel, filter paper, 4-methylumbelliferyl-β-D-cellobioside Kanokratana et al., 2008496 stable at pH 3−11 and maintains
BCC4124 β-D-cellobioside ∼50% at 70−90 °C for 30 min
Heterobasidion irreg- Cel7A 4.0 45 pNPL pNPL Momeni et al., 2013449
ulare TC 32-1
Humicola grisea var. Aspergillus ory- Exo1 5.0 65 pNPC Avicel, CMC, Glc2/Glc3/Glc4/Glc5/Glc6, p-nitrophenyl-β-D- Takashima et al., 1998497 stable at pH 3.0−10.0 at 4 °C for
thermoidea zae glucoside, pNPC 20 h
IFO9854
Humicola grisea var. Aspergillus ory- CBHI 5.0 60 pNPC Avicel, CMC, xylan, Glc2/Glc3/Glc4/Glc5/Glc6, p-nitro- Takashima et al., 1996,498 stable at pH 2.0−10.0 at 4 °C for 20
thermoidea zae phenyl-β-D-glucoside, pNPC Takashima et al., 1998497 h, and at 55 °C for 10 min
Review

IFO9854

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 Table 5. continued
temp
opt (°
organism expression host enzyme pH opt C) substrate for opt substrate specificity ref comments
Humicola grisea var. Aspergillus ory- EGL1 5.0 55−60 pNPC Avicel, CMC, xylan, Glc2/Glc3/Glc4/Glc5/Glc6, p-nitro- Takashima et al., 1996,498 stable at pH 5.0−11.0 at 4 °C for
thermoidea zae phenyl-β-D-glucoside, pNPC Takashima et al., 1998497 20 h, and at 60 °C for 10 min
IFO9854
Chemical Reviews

Humicola insolens Aspergillus ory- CBH1, 5.5 Glc3 PASC, Glc3/Glc4/ Glc5/Glc6, Avicel Schou et al., 1993,442 Schülein, assay conditions 37 °C
zae Cel7A 1997,499 Xu et al., 2009500
Humicola insolens Aspergillus ory- EG1, 5−6 CMC CMC, PASC, Glc3/Glc4/ Glc5/Glc6 pNPC, pNPL, Avicel, Schou et al., 1993,442 Schülein, assay conditions 40 °C; 35% residual
zae Cel7B BC, pretreated corn stover, xyloglucan, xylan, arabinox- 1997,499 Vlasenko et al., activity on CMC after 3 h at 60 °C
ylan, mannan, galactomannan 2010332 and pH 5.0
Irpex lacteus MC-2 Cel1, Ex-1 5.0 50 Avicel Avicel, CMC, BC, pNPL, pNPC Hamada et al., 1999501
Irpex lacteus MC-2 Cel2, Ex-2 5.0 50 Avicel Avicel, CMC, BC, pNPL, pNPC Hamada et al., 1999501
Melanocarpus albo- Cel7A 6.0 65−70 hydroxyethylcellulose hydroxyethylcellulose, 4-methylumbelliferyl-β-D-lactoside, Miettinen-Oinonen et al.,
myces CMC, Avicel, PASC 2004,502 Szijártó et al.,
2008503
Melanocarpus albo- Saccharomyces Cel7B 65 Avicel 4-methylumbelliferyl-β-D-lactoside, Avicel, CMC, hydroxye- Voutilainen et al., 2007,504 assay conditions pH 6.0
myces cerevisiae (cbh) thylcellulose, PASC, filter paper, 2-chloro-4-nitrophenol-β- Szijártó et al., 2008,503 Miet-
D-lactoside tinen-Oinonen et al., 2004,502
Voutilainen et al., 2009505
Myceliophthora ther- Pichia pastoris EG7A 5.0 60 CMC barley β-glucan, CMC, lichenan, arabinoxylan, xylan, filter Karnaouri et al., 2014506 stable at pH 3−11, retaining initial
mophila paper, hydroxyethylcellulose, Avicel, Glc3/Glc4/Glc5 activity after 24 h
Penicillium chrysoge- CBH1 pNPC Hou et al., 2007507
num FS010
Penicillium decum- CBHI, pNPC Gao et al., 2012508
bens Cel7A

1336
Penicillium decum- Saccharomyces Cel7B 4.0 60 CMC CMC, barley β-glucan, PASC, pNPC, Avicel, xylan Wei et al., 2010509 stable at pH 3−8 at 4 °C for 16 h;
bens cerevisiae maintained >90% initial activity
after 1 h at 60 °C
Penicillium occitanis CBHI 4−5 60 pNPC/PASC pNPC, pNPL, Avicel, filter paper, PASC, Glc3/Glc5 Limam et al., 1995510 stable at pH 2−9; maintains activity
Pol6 below 60 °C, but loses ∼50%
activity after 30 min at 60 °C, and
inactivated at 70 °C
Penicillium pulvillo- CBH1 4.2 Avicel Avicel, CMC, p-nitrophenyl-β-D-glucoside, glucuronoxylan Marjamaa et al. 2013511 assay conditions 45 °C
rum
Phanerochaete chrys- CBH62, Avicel, pNPL, pNPC, CMC Uzcategui et al., 1991512
osporium CBH1.1,
Cel7C
Phanerochaete chrys- Cbh58, Avicel, pNPL, pNPC, BMCC, hydroxyethylcellulose amor- Uzcategui et al., 1991,512 von
osporium CBH1.2, phous cellulose Ossowski et al. 2003461
Cel7D
Talaromyces emerso- Cbh1A, 4.1 66−69 2-chloro-4-nitrophenyl- Avicel; pNPC, pNPL, 2-chloro-4-nitrophenyl-β-D-cellobio- Tuohy et al., 2002513 pH optimum measured at 50 °C;
nii IMI 392299 CBH IB, β-D-cellobioside, side, 2-chloro-4-nitrophenol-β-D-cellotrioside, 2-chloro-4- temperature optimum measured at
(Rasamsonia Cel7A pNPL nitrophenol-β-D-lactoside, 4-methyl-umbelliferyl-β-D-cello- pH 5.0; T1/2 of 68 min at 80 °C
emersonii) trioside and pH 5.0
Talaromyces f unicu- XynA 3.5 55 barley β-glucan barley β-glucan, CMC, pNPL, pNPC, Glc3/Glc4/Glc5, xylan, Texier et al., 2012,514 Furniss et maintains >80% activity at pH 3−4.5
losus IMI 378536 arabinoxylan al., 2005515
[Penicillium f uni-
culosum]
Thermoascus auran- Trichoderma ree- Cel7A 5.0 65 4-methylumbelliferyl- 4-methylumbelliferyl-β-D-lactoside, 2-chloro-4-nitrophenol- Voutilainen et al., 2008381
tiacus ALKO4242 sei β-D-lactoside β-D-lactoside, Avicel, PASC
Thermoascus auran- Saccharomyces Cel7A 6.0 65 pNPL Avicel, PASC, pNPL, pNPC Hong et al., 2003516 stable at pH 3.0−9.0 at 40 °C for 24
Review

tiacus IFO 9748 cerevisiae h; maintained >80% initial activity

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after 1 h at 65 °C
 Table 5. continued
temp
opt (°
organism expression host enzyme pH opt C) substrate for opt substrate specificity ref comments
Thielavia australien- Cel7A Avicel, PASC Xu et al., 2009500
sis
Thielavia terrestris Cel7A Avicel, PASC Xu et al., 2009500
Chemical Reviews

Thielavia terrestris Cel7C PASC, Avicel, BC, pretreated corn stover, CMC, xyloglucan, Vlasenko et al., 2010332 assay conditions 50 °C and pH 5.0
xylan, arabinoxylan, mannan, galactomannan
Trichoderma harzia- CBHI, 5.0 50 pNPC BMCC, Avicel, Sigmacell 20, CMC, pNPC, cNPL Colussi et al., 2011,517 Textor
num FP 108/ Cel7A et al., 2013466
IOC-3844
Trichoderma longi- Aspergillus nidu- Egl1, 4.5 50 CMC CMC, barley β-glucan, laminarin, xylan Ganga et al., 1997518 narrow pH activity (35% activity on
brachiatum lans Cel7A CMC at pH 4 and 5.5); opt on
CECT 2606 xylan at pH 4.5 and 60 °C
Trichoderma pseu- Pichia pastoris Cel7B 45 4-methylumbelliferyl- 4-methylumbelliferyl-β-D-lactoside, CMC Mitrovic et al., 2014519 assay conditions pH 4.8
dokoningii β-D-lactoside
Trichoderma reesei Saccharomyces EGI, Cel7B 60 PASC CMC, Glc3/Glc4/Glc5/Glc6, pNPC, pNPL, PASC, Avicel, Van Arsdell et al., 1987,330 assay conditions pH 5.0; 50%
cerevisiae BC, pretreated corn stover, CMC, xyloglucan, xylan, Biely et al., 1991,331 Bailey et residual activity on CMC after 3 h
arabinoxylan, mannan, galactomannan, barley β-glucan, al., 1999,286 Vlasenko et al., at 60 °C and pH 5.0
hydroxyethylcellulose 2010332
Trichoderma reesei CBHI, 4.5 65 4-methylumbelliferyl- 4-methylumbelliferyl-β-D-lactoside, 2-chloro-4-nitrophenol- Boer and Koivula, 2003,327
L27 Cel7A β-D-lactoside β-D-lactoside, 3,4- dinitrophenyl-β-D-cellobioside, 3,4- Becker et al., 2001328
dinitrophenyl-β-D-lactoside, BMCC
Trichoderma viride EGI 5.0 50 CMC CMC, Glc3 Kwon et al., 1999 retains >80% activity at pH 3.5−6.0
HK-75
Trichoderma viride Saccharomyces CBHI 5.8 60 CMC CMC Song et al., 2010520 retains >70% of maximal activity at

1337
AS 3.3711 cerevisiae pH 3.8−7.0 and 60% of maximal
activity at 40−90 °C
Review

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Chemical Reviews Review

Table 6. GH7 Rate Constantsa

isolated CBH CBH plus EG
ref property value substrate property value subtrate ref
−1
476 kcat (s ) 7.1 ± 3.9 Iα
564 kcat (s−1) 6.8 ± 3.5 III
307 kcat (s−1) 2.8 ± 0.4 BC
557 kcat (s−1) 2.2 ± 0.5 BC
561 kcat (s−1) 2.4 BMCC kcat (s−1) 1.5 ± 0.2 BC 557
470 kcat,gly (s−1) 0.415 Glc8
441 kcat,gly (s−1) 10.8 Glc9
564 koff (s−1) 0.20 ± 0.01 III
307 koff (s−1) 0.0007 ± 0.1 BC
561 koff (s−1) 0.01 BMCC
307 Papp 61 ± 14 BC
557 Papp 66 ± 7 BC Papp 50 ± 3 BC 557
561 Papp 22.6 BMCC
557 kobs (s−1) 0.1 ± 0.05 BC kobs (s−1) 1.45 ± 0.5 BC 557
a
Advanced experimental techniques have allowed for the determination of various rate constants in the processive cycle of GH7 CBHs. In each case,
the CBH is intact TrCel7A, and the EG is TrCel5A. Substrate abbreviations not previously defined are III = cellulose III, Iα = cellulose Iα, Glc8 =
cellooctaose, and Glc9 = cellononaose. The rate constant kcat represents the rate constant for the complete inner processive cycle that includes
processivity, hydrolysis, and product expulsion, whereas kcat,gly specifically describes the barrier for the glycosylation reaction and was computed via
advanced molecular simulation techniques. Papp was called n in the original publication by Cruys-Bagger et al.561 Rate constant kobs is calculated from
the rate of cellobiose produced normalized by the concentration of CBH with occupied active site.557

Abuja et al. SAXS study from 1989 wherein the addition of xylan
to TrCel7A was demonstrated to stiffen the linker.401
It has been known for some time that modifications to
cellulase linkers modify the enzyme activity, typically in
detrimental ways when major changes are made.251,405 On the
basis of pioneering work using SAXS and other biophysical
methods, we now know that cellulase linkers such as those
found in fungi are intrinsically disordered pro-
teins.407,408,414,416,417 For cellulases from T. reesei, linker glycan
patterns are either known or at least the range of mannose
residues have been characterized on linkers.273,368,418−420 New
bioinformatics analyses have emerged recently that suggest a
more general means to analyze linkers beyond sequence
alignments, yielding more detailed quantitative information
about linker function and optimization. 417 Lastly, new
theoretical analysis coupled with biophysical measurements
has suggested that glycosylated linkers play a direct role in
cellulose binding, similar to the CBM but with a nonspecific,
dynamic role.393 However, despite these strides, key questions
remain regarding the detailed mechanistic roles of cellulase
linkers. For example, it is unknown why cellulases from different
families employ linkers of different average lengths, or why and
how linkers from different organisms affect overall enzyme
stability and activity.290 Certainly glycosylation is known to be
important for linker protection404 and more recently for binding
to cellulose,393 but glycosylation is known to be quite
heterogeneous and it can differ significantly between fungi.421
How linker sequences and glycan patterns coevolved is an open
question. Moreover, most fungal cellulase linkers have been
examined from cellulases from T. reesei or similar fungi.
However, some fungal cellulases, such as those found in
rumen fungi employ linkers with dramatically different sequence
characteristics such as with extremely high asparagine
content.417 Clearly, many structure−function studies on both
CBMs and linkers remain yet to be done to fully understand
their detailed roles in cellulose deconstruction.
1338
6. FAMILY 7 GLYCOSIDE HYDROLASES
GH7 enzymes are commonly among the most prevalent
cellulolytic enzymes in secretomes of biomass-degrading fungi,
almost undoubtedly because the processive GH7 cellulases
provide the majority of hydrolytic turnover during fungal
cellulose depolymerization. For example, T. reesei secretes a
single GH7 CBH and a single GH7 EG.41 White-rot
basidiomycete fungi, such as the model fungus P. chrysosporium,
degrade lignin in plant cell walls most likely for enhanced access
to biomass polysaccharides, and also employ GH7 CBHs and
EGs for cellulolytic action.426,427 Similar to some organisms that
employ GH7 CBHs to degrade biomass, P. chrysosporium has
multiple GH7 CBHs, and in virtually all organisms that exhibit
multiple GH7 CBH genes, the need for multiple, similar CBHs
from the same family is as of yet unknown. Moreover, unlike
other GH enzymes in this review (i.e., GH6, GH5, GH12, and
GH45), GH7 enzymes have not been found in bacteria or
archaea to date, but mainly have been found in fungi. The CAZy
database currently lists nearly 5000 GH7 sequences, although
some are noted to be gene fragments only, not full-length
enzymes.151−153 Quite recently, GH7 enzymes have been
characterized in crustaceans,428 protist symbionts,429 strameno-
piles,428 and slime molds (or social amoeba),430−432 demon-
strating that they are not only found in fungi. Interestingly, as
highlighted by King et al., these nonfungal GH7 cellulases offer
distinct evolutionary branches from fungal cellulases, inspiring
the study of the similarities and differences with their fungal
counterparts.383,428,432,433
The first discovered and characterized GH7 cellulase was
characterized originally from T. reesei in the late 1970s and early
1980s, which was originally denoted CBH I.434−438 As
mentioned in section 4, the gene was subsequently sequenced
simultaneously by two groups in the early 1980s.281,282 Initial
work from Pettersson et al. also discovered that TrCel7A was a
multimodular protein, which were among the first studies to
discover the coupling of binding and catalytic function in
cellulases, as discussed above.343,344 As a result of the significant
body of work on GH7 cellulases, especially CBHI from T. reesei
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Chemical Reviews Review

Table 7. Reported Fungal GH7 Crystal Structures

source and original name in PDB resolution
primary citation code (Å) brief highlights ref
CBH Structures
Trichoderma reesei CBH1/Cel7A 1CEL 1.80 first GH7 structure reported; complex with o-iodobenzyl-1-thio-β-D-cellobioside. 172
1DY4 1.90 complex with (S)-propranolol 463
1EGN 1.60 engineered variant E223S/A224H/L225 V/T226A/D262G 328
1Q2B 1.60 engineered variant D241C/D249C 461
1Q2E 1.75 engineered variant, deletion of residues 245−252; complex with Glc2-S-Glc2 461
2V3I 1.05 highest resolution of a CBH1 from T. reesei; complex with (R)-dihydroxy unpublished
phenanthrenolol.
2CEL 2.00 active-site mutant E212Q 446
3CEL 2.00 active-site mutant E212Q; complex with cellobiose 446
4CEL 2.20 active-site mutant D214N 446
5CEL 1.90 active-site mutant E212Q; complex with two cellotetraose molecules 173
6CEL 1.70 active-site mutant E212Q; complex with cellopentaose and cellotetraose 173
7CEL 1.90 active-site mutant E217Q; complex with cellohexaose and cellobiose 173
4C4C 1.45 active-site mutant E217Q; Michaelis complex 441
4C4D 1.32 active-site mutant E217Q; covalent glycosyl-enzyme intermediate trapped using DNP-2- 441
deoxy-2-fluoro-cellotrioside
Heterobasidion irregulare Cel7A 2YG1 1.90 449
2XSP 1.70 complex with xylose 449
Limnoria quadripunctata Cel7B 4GWA 1.60 first structure of a nonfungal GH7 CBH from a salt tolerant marine animal 383
4HAP 1.60 complex with cellobiose 383
4HAQ 1.90 complex with cellobiose and cellotriose 383
4IPM 1.14 complex with thiocellobiose 383
Melanocarpus albomyces Cel7B 2RFW 1.60 thermotolerant GH7 CBH1 465
2RFY 1.70 complex with cellobiose 465
2RFZ 1.80 complex with cellotriose 465
2RG0 2.10 complex with cellotetraose 465
Phanerochaete chrysosporium 1GPI 1.32 first structure of a GH7 CBH1 from a basidiomycete 460
Cel7D
1H46 1.52 complex with (R)-propranolol 462
1Z3T 1.70 complex with cellobiose 458
1Z3V 1.61 complex with lactose 458
1Z3W 1.70 complex with cellobioimidazole 458
Talaromyces emersonii Cel7A 1Q9H 2.35 first structure of a thermostable GH7 CBH 464
3PFJ 1.36 unpublished
3PFX 1.26 complex with cellobiose unpublished
3PFZ 1.10 complex with cellotetraose unpublished
3PL3 1.18 complex with cellopentaose unpublished
Trichoderma harzianum Cel7A 2Y9N 2.89 unpublished
2YOK 1.67 466
EG Structures
Trichoderma reesei Cel7B 1EG1 3.60 450
Fusarium oxysporum Cel7B 1OVW 2.70 first GH7 EG; complex with thiocellotriose 174
2OVW 2.30 complex with cellobiose 448
3OVW 2.30 448
4OVW 2.30 complex with epoxybutyl cellobiose 448
Humicola insolens Cel7B 1A39 2.20 engineered variant S37W/P39W 457
2A39 2.20 wild-type 452
1DYM 1.75 engineered variant E197A 452
1OJI 2.15 engineered variant E197S 653
1OJJ 1.40 engineered variant E197S; complex with lactose 653
1OJK 1.50 engineered variant E197S; complex with cellobiose 653

in the late 1970s and 1980s, these enzymes were included in the
first classification of cellulases from Henrissat et al., denoted as
family C enzymes based on hydrophobic cluster analysis.439
In this section, we review the history of GH7 studies primarily
starting from the first structural report in 1994.172 As these
enzymes have been the focus of many studies, given their
abundance in natural biomass degrading fungi and importance
1339
in industrial biomass conversion, a significant number of studies
have been applied to elucidate the GH7 catalytic mechanism,
understand the basis of GH7 CBH processivity, improve their
thermostability and activity, and ascertain other important
features of their structure and function. Here, we focus on
structure and activity studies of these key enzymes. We note that
we do not include a significant review of studies before the first
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Chemical Reviews Review

Figure 22. Crystal structure of the first GH7 CBH and EG. The ligand from the TrCel7A Michaelis complex (PDB code 4C4C441) is shown in all
panels. (A) CBH TrCel7A CD (PDB code 1CEL172) view from side, exhibiting the β sandwich structure that is characteristic of GH7 enzymes.
TrCel7A was the first GH7 structure solved and is the best-characterized member of GH7. (B) TrCel7A view from bottom showing the more closed
substrate binding “tunnel”. (C) EG F. oxysporum Cel7B (PDB code 1OVW174) view from side. (D) FoCel7B view from the bottom showing the more
open binding “groove”. (E) TrCel7A Michaelis complex (PDB code 4C4C441) exemplifies the standard numbering of the substrate binding sites
(catalytic residues shown in green for reference). A cellulose chain enters from the −7 site. Hydrolysis occurs between the −1 and +1 sites; thus, the
+1/+2 sites are termed the “product sites”.

structural reports, except where needed, as these studies have
been reviewed extensively.32,316,440 We note that, despite the
huge body of work conducted to date on these enzymes, there
are still major elements of their function that remain to be
elucidated, especially related to improving their stability and
activity. Moreover, given the wealth of GH7 sequences available
from genomics and metagenomics efforts, expression and testing
1340
of GH7 gene products are now of acute importance for the
continued development of structure−activity relationships. A
summary of GH7 structures that are discussed are provided in
Table 7, and biochemical data for characterized GH7s are
provided in Table 5.
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Chemical Reviews
6.1. Structural Studies and Catalytic Function
6.1.1. TrCel7A: Wild-Type. The first crystal structure of a
GH7 member was presented in 1994 (PDB code 1CEL),
revealing the structure of the catalytic core of the CBH
TrCel7A,172 whose primary structural characteristic is a large β-
sandwich formed by two large antiparallel β-sheets. This original
structure was a complex with inhibitor o-iodobenzyl-1-thio-β-D-
cellobioside; Figure 22A,B shows the 1CEL crystal structure
with the cellononaose ligand from the solved structure of the
Michaelis complex (PDB code 4C4C441), published 20 years
later. The structure revealed a binding tunnel that was estimated
to have 7 binding sites, roughly twice as long as that of the
TrCel6A CBH (the only other CBH with a solved structure at
that time192). Like Cel6A, the binding tunnel was lined with
tryptophan residues (three in Cel6A and four in Cel7A).
Though it was previously determined via 1H NMR that TrCel7A
(later generalized to the entire GH7 family442) employs a two-
step, retaining catalytic mechanism (contrasted with the one-
step, inverting mechanism of TrCel6A),443 the structural
machinery had not yet been revealed. This mechanism utilizes
two glutamate residues that generally reside approximately 5.5 Å
apart in the catalytically active conformation (Figure 23).444
Figure 23. First structural picture of a GH7 active site. TrCel7A (PDB
code 1CEL) was the first GH7 structure solved. The o-iodobenzyl-1-
thio-β-D-cellobioside inhibitor captured in the product sites helped to
identify Glu217 as the potential acid/base in the retaining mechanism.
Glu212 was proposed as the nucleophile with the role of Asp214 not
yet elucidated.
One of these residues is the nucleophile for the first step
(glycosylation), and the other is the acid/base. The acid/base
donates a proton to the glycosidic oxygen in the first step and
removes a proton from a water molecule that serves as the
second step nucleophile. On the basis of the 1CEL crystal
structure, two glutamate residues were proposed as the catalytic
residues: Glu217 as the catalytic acid/base and Glu212 as the
nucleophile. On the basis of its proximity to the O4 atom of the
o-iodobenzyl-1-thio-β-D-cellobioside glucosyl moiety (occupy-
ing the putative position of the cleavable glycosidic bond),
Glu217 was suggested as the acid/base (Figure 23).172 A third
acidic residue, Asp214 in TrCel7A, was noted to potentially be
involved in the chemical steps172 due to its proximity to the
active site and close contact with the putative nucleophile,
Glu212. In addition, the location of the proposed active site
suggested that the cellulose chain is cleaved from its reducing
end. This was consistent with previous findings that established
the preference of TrCel7A to hydrolyze the reducing ends of
1341
Review
cellulose chains using cello-oligosaccharides with tritium-labeled
reducing ends.445 It was also proposed that the longer binding
tunnel, along with the location of cleavage that is skewed toward
the tunnel exit, would make Cel7A more processive than Cel6A.
Given its importance as the first structural representative of a
GH7 CBH and the fact that many studies have used this enzyme
as the model GH7 CBH, in the discussion that follows, the
numbering of individual enzyme residues corresponds to that of
TrCel7A, unless otherwise noted.
6.1.2. TrCel7A Catalytic Mutants. Site-directed muta-
genesis of TrCel7A was subsequently employed in 1996 to
further probe the roles of the triad of acidic residues in glycosidic
bond cleavage by mutation to their isosteric amide counter-
parts.446 The catalytic activity of the individual point mutants
E212Q, D214N, and E217Q was impaired on 2-chloro-4-
nitrophenol-β-D-lactoside, with kcat reductions of 1/2000, 1/85,
and 1/370, respectively, compared to the wild-type. In addition,
E212Q and E217Q mutants lost all catalytic activity on
crystalline cellulose.446 Crystal structures of each mutant
showed that the active site architecture and overall fold of the
protein are identical in all the mutants and the wild-type, thus
confirming that the activity loss was due to the catalytic roles of
these three residues (PDB codes 2CEL, 3CEL, and 4CEL).
Importantly, the D214N structure (PDB code 4CEL) revealed a
calcium ion bound to Glu212, supporting the hypothesis that
this residue is the charged species in the precatalytic state.
6.1.3. F. oxysporum Cel7B with Active Site-Spanning
Nonhydrolyzable Inhibitor. Also in 1996, important
structural details of the substrate at the active site during
catalysis were revealed by the first crystal structure of a GH7 EG
from F. oxysporum (FoCel7B) complexed with a nonhydrolyz-
able thiooligosaccharide inhibitor (PDB code 1OVW, Figure
22C,D).174 EGI was previously characterized as having four
binding sites via analytical HPLC measurements and kinetic
assays of this enzyme on reduced cello-oligomers of varying
length (DP 3−6).442 The active site-spanning substrate
analogue captured in the FoCel7B crystal structure occupies
the −2, −1, and +1 binding sites (Figure 24). Along with the
structure of a chitin-degrading enzyme published earlier the
same year,447 this crystal structure was the first to reveal an
Figure 24. Enzymatic substrate distortion in the GH7 active site.
FoCel7B (PDB code 1OVW174) was the first GH7 EG structure solved.
Note the ring distortion at the −1 subsite that is midway between a 4E
envelope and a 1,4B boat conformation. Sulfur atoms (shown in yellow)
replace the glycosidic oxygen atoms. Glu197 is the nucleophile, and
Glu202 is the catalytic acid/base.
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Figure 25. Loop structures in GH7 enzymes. The major loops of TrCel7A (PDB code 4C4C441) and TrCel7B (PDB code 1EG1450). The loop
nomenclature is taken from Momeni et al.449 Note the deletions of most major loops in TrCel7B. The ligand from 4C4C is shown in both panels.

Figure 26. Key structural differences among GH7 EGs. (A) At the tunnel entrance, TrCel7B (PDB code 1EG1, shown in orange) is the only GH7 EG
with solved structure to naturally maintain the tryptophan stacking seen in TrCel7A. Also shown is the HiCel7B S37W/P39W mutant (PDB code
2A39, magenta). (B) Similar to the tunnel entrance, TrCel7B is the only of the three GH7 EGs with solved structure to maintain the aromatic stacking
(Tyr38) at the −4 site seen in TrCel7A (Trp38). The HiCel7B S37W/P39W mutant is also shown, as well as the residues occupying this site in
FoCel7B (PDB code 1OVW, gray) and wild-type HiCel7B (PDB code 2A39, slate): Ile37 and Ser37, respectively. (C) The product binding sites are
quite different in the three EGs as compared with TrCel7A. Two of the three arginine residues that contact the ligand in TrCel7A are absent in all three
EGs (Arg251 and Arg394). Arg267, however, is found in HiCel7B and FoCel7B. These two EGs also have an additional residue that hydrogen bonds
to the substate (His209) that is not found in either of the T. reesei enzymes. In all panels, the background protein from TrCel7B (transparent orange
“cartoon”) and the ligand from the TrCel7A Michaelis complex (PDB code 4C4C, in aquamarine “sticks”) are shown.

intact glycosidic bond across the cleavage sites (Figure 24).190
The glucosyl ring at the −1 subsite was distorted into a nonchair
conformation (later termed a “skew boat”448). As discussed in
section 3.2, GHs have been proposed to distort substrate ring
conformations to aid catalytic bond cleavage. This structure was
further evidence that this distortion is indeed critical for
providing the nucleophile access to the −1 anomeric carbon for
nucleophilic attack.174 The structure of FoCel7B also provided
additional structural evidence confirming the importance of the
three acidic residues that had been identified previously for
TrCel7A. Glu202 (corresponding to TrCel7A Glu217) hydro-
gen bonds (at an O-S distance of 2.7 Å) to the glycosidic sulfur
of the inhibitor between the −1 and +1 glucosyl rings (Figure
24). In addition, Glu197 (corresponding to TrCel7A Glu212)
was poised for nucleophilic attack, residing 3.2 Å away from the
−1 anomeric carbon. Finally, it was noted that Asp199
(corresponding to TrCel7A Asp214) formed a hydrogen bond
(2.5 Å) to the catalytic nucleophile. Though its catalytic role was
still unclear, it was speculated to be involved in “proton
1342
shuffling” around the active site, maintaining the catalytically
active protonation states for the nucleophile and acid/base.
Though not discussed at length in the original publication, this
structure was also the first to reveal the dramatic loop
shortenings that were later identified as being characteristic of
GH7 EGs. In fact, comparison with the structure of TrCel7A
shows that loops B1, B2, B3, B4, A1, and A4 are all considerably
shortened or absent altogether in FoCel7B. Figure 25 provides
the loop nomenclature utilized herein for GH7 cellulases,
following Momeni et al.449
6.1.4. TrCel7B. In 1997, the crystal structure of T. reesei EG
(PDB code 1EG1), at the time defined as EGI but later renamed
to TrCel7B, revealed the same overall fold as TrCel7A but with a
completely open binding site “cleft” compared to the tunnel
observed for TrCel7A.450 In TrCel7B, loops B2, B3, B4, and A4
are absent (Figure 25). However, loop A1 is of similar length in
TrCel7B as it is in TrCel7A, though slightly more open in the
crystal structure. Sequence alignments had previously indicated
that major deletions seen in TrCel7B mapped to the tunnel-
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Figure 27. Comparison of the loops of GH7 EGs. Compared with GH7 CBHs, GH7 EGs display significantly shorter (or altogether deleted) loops
that connect the two faces of the β sandwich. (A) TrCel7B (PDB code 1EG1) displays the most truncated loop structures of any GH7 cellulase with a
solved structure. (B) FoCel7B (PDB 1OVW) and (C) HiCel7B (PDB code 2A39) have slightly more prominent B3 and B4 loops than TrCel7B. In all
panels, the ligand from the TrCel7A Michaelis complex (PDB code 4C4C) is shown in aquamarine “sticks”.
forming loop regions of TrCel7A. The TrCel7B crystal structure
confirmed the absence of these loops, yielding a more open
binding cleft. These differences in loop structure potentially
provided a strong rationale for the difference in functionality of
CBHs versus EGs. The additional surface loops in CBHs were
hypothesized to prevent an extracted cellulose chain from
readhering to the crystalline surface postcatalysis as well as
keeping the chain threaded in the tunnel for multiple hydrolytic
events before dissociation enabling CBHs to processively cleave
cellobiose much more effectively than EGs.
The same year, the identity of the GH7 catalytic nucleophile
was confirmed by isolating the glycosyl-enzyme intermediate of
FoCel7B.451 The glycosyl-enzyme intermediate was captured by
incubation of the enzyme with 2′,4′-dinitrophenyl 2-deoxy-2-
fluoro-β-cellobioside, a method that has found great utility in
identifying active site residues, as mentioned in section
3.2.170,206,452−454 This class of inhibitors slows both steps of
the retaining mechanism due to the presence of the C2 fluorine.
Coupling this with a good leaving group that accelerates only
the first step (glycosylation) allows for “trapping” of the
glycosyl-enzyme intermediate.202,451,455 Subsequent to trapping,
characterization via mass spectrometry allows for identification
of the nucleophilic residue. In this case, the nucleophile was
identified as Glu197, which is completely conserved in GH7s
and corresponds to Glu212 in TrCel7A. Around the same time,
a combination of comparative liquid chromatography coupled
online to electrospray ionization mass spectrometry, tandem
mass spectrometry, and microsequencing applied to TrCel7A
bound with an epoxide-based inhibitor provided direct
experimental proof for Glu212 as the catalytic nucleophile for
that enzyme and also revealed the glycosylation pattern of the
core protein.456
6.1.5. H. insolens Cel7B S37W/P39W Mutant. Also in
1997, the structure of a double mutant of H. insolens Cel7B
(HiCel7B; PDB code 1A39) was published wherein additional
sugar-binding sites were engineered on the basis of comparison
with TrCel7A.457 Two tryptophan residues were inserted
(S37W and P39W) to introduce “+3” and “+4” binding sites
(Figure 26A,B). The resulting mutant maintained wild-type
level of activity on soluble substrates, but had a slightly
decreased Michaelis constant KM (by 30%) on PASC, indicating
slightly higher binding on longer substrates. Subsequent
publication of the wild-type structure of this enzyme (PDB
code 2A39)452 further described the more open binding cleft of
EGs first observed structurally for FoCel7B and first discussed in
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detail for TrCel7B (Figure 27).450 These findings also solidified
the structural basis for the observed differences in EGs and
CBHs. The same study that presented wild-type HiCel7B also
utilized site-directed mutagenesis to confirm the catalytic roles
of Glu202 (acid/base, corresponding to Glu217 in TrCel7A)
and Glu197 (nucleophile, corresponding to Glu212 in
TrCel7A).452 E197A and E202A mutants had no detectable
catalytic activity, either on reduced oligosaccharides or p-
nitrophenylcellobioside substrates.452 This study also confirmed
the identity of the catalytic nucleophile for HiCel7B utilizing the
technique previously applied to FoCel7B451 in which the
glycosyl-enzyme intermediate is trapped and subsequently
characterized by mass spectroscopy.
The three EG structures described directly above constitute
the only GH7 EG structures solved to date. The most dramatic
structural difference between these EGs and TrCel7A is in the
loops that protrude from the two faces of the β sandwich. All of
the major loops (with the exception of A1 at the tunnel
entrance) are shortest in TrCel7B compared with the other
GH7 EGs; thus, its binding cleft is the most open GH7 cellulase
characterized to date (Figure 25B and Figure 27A). The
shortenings/deletions of both the so-called “exo” loop (B3) as
well as another large loop (B2) are particularly dramatic in
TrCel7B. However, compared with HiCel7B and FoCel7B,
TrCel7B has the longest entrance site loop (A1), which is on par
with that of TrCel7A. Further comparing HiCel7B with
FoCel7B shows that the loop structures for these two enzymes
are nearly identical, with the minor exception of a slightly
lengthened loop in HiCel7B on the back of the binding tunnel
entrance.
All three GH7 EGs align remarkably well to TrCel7A with
regards to active site protein residues (Figure 28). The
aromatic−carbohydrate interactions (discussed in more detail
below) are conserved in all three EGs at Trp367 (over the −2
subsite) and Trp376 (over the +1 subsite). However, the
important interaction between Trp40 and the −7 sugar residue
is naturally present only in only one known EG structure,
TrCel7B (Figure 26A). TrCel7B is also the only EG to naturally
maintain an aromatic stacking interaction at the −4 subsite,
albeit with a tyrosine rather than the tryptophan found in
TrCel7A (both at residue number 38, Figure 26B). The other
two EGs have no aromatic interaction here; this site is occupied
by isoleucine in FoCel7B and by serine in HiCel7B.
Despite the exceptional similarity in catalytic machinery and
some similarity in aromatic−carbohydrate interactions, signifi-
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Figure 28. Sequence alignment of major GH7 enzymes. Sequence alignment of three GH7 CBHs (TrCel7A, PcCel7D, and HirCel7A) and three EGs
(TrCel7B, HiCel7B, and FoCel7B). Strictly conserved residues are shown in red block, and chemically similar residues in red text. The blue boxes
indicate chemical similarity across a grouping of residues. The secondary structural elements and residue numbering of TrCel7A are shown above the
sequences. Loop structures (A1, B1, etc.) are shown in black boxes. The catalytic triad is denoted by yellow stars. The sequence alignment was
generated with ESPript (http://espript.ibcp.fr).347

cant differences exist not only in the aforementioned loop
morphologies, but also in relevant enzyme residues at the tunnel
entrance and the product binding sites. As noted above,
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amongst EGs only TrCel7B maintains the protein−carbohy-
drate stacking between Trp40 and the −7 subsite sugar at the
tunnel entrance. Though both enzymes lack this interaction,
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Figure 29. Comparison of the loops of GH7 CBHs. The loops that extend from the faces of the β sandwich in GH7 CBHs enclose the substrate
binding tunnel to varying degrees. Among GH7 CBHs with known structures, the A2, A3, A4, B1, B2, and B4 loops are fairly similar. (A) TrCel7A
encloses substrate most fully of any GH7 CBH. (B) PcCel7D features a shortening of the A1 loop and a natural deletion of six residues on loop B3
(“exo” loop) that give it a more open active site than TrCel7A (Figure 30A). (C) HirCel7A features a lengthened A1 loop (Figure 30A) and a slightly
shortened B3 loop (compared with TrCel7A) due to the natural deletion of two residues. In all panels, the ligand from the TrCel7A Michaelis complex
(PDB code 4C4C) is shown in aquamarine “sticks”.
HiCel7B and FoCel7B are not identical at the entrance, as
HiCel7B has essentially no interactions with the ligand here, but
FoCel7B has an arginine residue (Arg41, FoCel7B numbering)
within hydrogen bonding distance to both the −7 and −6
subsites that TrCel7A lacks. The Asn49 residue in TrCel7A
forms hydrogen bonds to both the −7 and −6 subsite glucosyl
moieties, which is not found in any of the GH7 EGs.
Moving toward the tunnel exit, an increasing number of
discrepancies are present between TrCel7A and the three GH7
EGs. This is partly due to missing loops that enclose the tunnel
exit in TrCel7A (A4 and B4, Figure 25). Another key difference
in the product sites is three arginine residues present in TrCel7A
(Arg 251, Arg267, and Arg394) that are at or close to hydrogen
bonding distance to the +1/+2 glucosyl residues (Figure 26C).
Arg251 is located at the base of loop B3 and has been identified
as being particularly important to product coordination.458 The
guanido group of this residue has been observed to make direct
hydrogen bonds with the O5 and O6 atoms of the sugar in the
+1 binding site.458 All three EGs lack this residue. All three also
lack Arg394, which has been identified in a computational study
of processivity in TrCel7A as being one of the key drivers of
processive motion.459 The conservation of these arginine
residues in processive cellobiohydrolases (Figure 28) and
absence in relatively nonprocessive EGs perhaps affirm their
importance in driving processive motion. In contrast to the
other two arginine residues present at the TrCel7A product
sites, HiCel7B and FoCel7B, maintain the Arg267 interaction.
These latter two EGs also have an additional interaction at the
product sites that both T. reesei enzymes lack: His209, which is
within hydrogen bonding distance to the C2 hydroxyl of the +1
glucosyl residue.
6.1.6. TrCel7A: Cello-Oligomer Complexes. The exten-
sive insights into GH7 architecture provided by the three EG
structures were greatly enhanced by subsequent publication of
new CBH structures. For example, crystal structures of TrCel7A
E212Q and E217Q mutants published in 1998 gave much
greater insight into the binding of cello-oligomers in GH7
CBHs.173 These structures included the binding of (1) two
cellotetraose molecules on either side of the vacant −3 site
(PDB code 5CEL), (2) cellopentaose in −6 to −2 and
cellotetraose in +1 to +4 (PDB code 6CEL), and (3)
cellohexaose in −7 to −2 and cellobiose in +1/+2 (PDB code
7CEL). This more complete view of cellulose chain binding
revealed 9−10 binding sites (−7 to either +2 or +3) in a 50 Å
1345
Review
long tunnel (the +3 site has almost no interaction with
enzymatic residues, and the +4 site is outside of the binding
tunnel and has no carbohydrate−protein interactions). From −7
to −4 the chain binding for all ligands overlaps almost perfectly.
Two twists occur between −4 and −2 that essentially turn the
cellulose chain upside down. By linking the cellohexaose and
cellobiose contained in 7CEL and modeling a “skew boat”
configuration in the −1 site (based on previously published
chitobiase447 and FoCel7B174 structures), a theoretical model of
the Michaelis complex (PDB code 8CEL) was also presented
that became the basis for modeling studies of this enzyme for the
next 15 years.
6.1.7. P. chrysosporium Cel7D. The white-rot basidiomy-
cete P. chrysosporium secretes six unique GH7 CBHs.426 The
year 2001 marked the publication of the crystal structure of
PcCel7D, the first GH7 CBH crystal structure from a
basidiomycete,460 and revealed a substrate binding “groove”
that is more open than TrCel7A, but not as open as the GH7
EGs. This more open architecture is the result of several loop
deletions/shortenings compared with TrCel7A. At the binding
tunnel entrance, PcCel7D has a shortened A1 loop but also an
extra tyrosine (Tyr47) covering the entrance on the opposite of
the tunnel entrance that TrCel7A lacks (Figures 29 and 30A). In
addition, PcCel7D has a much shorter B3 loop and slightly
shorter B2 loop compared with TrCel7A. These loop variations
give a more accessible active site and may be the structural
explanation for PcCel7D’s enhanced kcat and Km on small soluble
substrates. The more open structure may also explain the
reduction in the binding of the cellobiose product, easing
product inhibition for this enzyme relative to TrCel7A.461 The
specific residues responsible for this are discussed further in
section 6.3.
The difference in substrate binding architecture between
TrCel7A and PcCel7D may also be relevant to the binding of
different enantiomers of the β-blocker propranolol. Both
enzymes prefer the S enantiomer over the R,462 yet crystal
structures of TrCel7A (PDB code 1DY4) could only be
obtained with S,463 while PcCel7D (PDB code 1H46) only gave
crystals with R.462 The enantioselectivity of TrCel7A may be a
largely entropy-driven process, as it has been shown to increase
with temperature.462 Thus, the more open active site, and the
resulting increase in solvation, could explain this difference.
6.1.8. TrCel7A: Exo Loop Engineering. The comparison
of TrCel7A and PcCel7D was extended in 2003 by extensive
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Figure 30. Key structural differences among GH7 CBHs. (A) GH7 enzymes exhibit A1 loops that are shortened (exemplified by PcCel7D in light blue,
PDB code 1GPI) and lengthened (exemplified by HirCel7A in light pink, PDB code 2YG1) compared with TrCel7A (green, PDB code 4C4C). On
the opposite side of the glucan chain, Trp40 is conserved in all cases. HirCel7A and PcCel7D both have an extra tyrosine that protrudes over the tunnel
entrance: HirCel7A Tyr101 over the top and PcCel7D Tyr47 on the opposite side. (B) Variation in tyrosines on the A3 and B3 loops in GH7 CBHs:
the lack of Tyr371 (numbering for TrCel7A) renders the B3 loop of ThCel7A (magenta, PDB code 2Y9L) more flexible, and LqCel7A (yellow, PDB
code 4GWA) lacks the Tyr247 equivalent and has a slightly shortened B3 loop; TrCel7A possesses a tyrosine at the tip of both the A3 and B3 loops,
and these have multiple conformation (PDB code 1CEL shown in teal, exemplifies this alternate conformation). (C) Key variable residues that
coordinate the glucosyl residues in the product sites. TrCel7A lacks the aspartate interaction at the +2 site that is seen in PcCel7D (Asp336) and
HirCel7A (Asp347). All three CBHs maintain the three arginine residues shown. In addition, due to the shortening of the B3 loop in PcCel7D (six
residues) and HirCel7A (two residues), both of these lack Tyr247 and Thr246, which are possessed by TrCel7A. In all panels, the ligand from the
TrCel7A Michaelis complex is shown in aquamarine “sticks” (PDB code 4C4C).

engineering of the active-site loop B3 (referred to as the “exo”
loop in the original publication) of TrCel7A; this loop is the
most prominent active-site structural difference between these
two enzymes.461 This included (separately) the introduction of
a Y247F mutation at the tip of the loop (no crystal structure
presented), deletion of the middle eight residues of the loop
(PDB code 1Q2E), and stabilization by introduction of a
disulfide bridge (PDB code 1Q2B). The three mutations
showed little effect on the hydrolysis of small, soluble substrates.
The deletion mutant gave enhanced activity on amorphous
cellulose, but a 50% activity reduction on crystalline cellulose;
this was associated with a reduction in processive character. The
disulfide bridge resulted in enhanced activity on both
amorphous and crystalline cellulose. Taken together, these
data confirmed the previous hypothesis that the B3 loop is
integral to the high processivity of TrCel7A.
6.1.9. TeCel7A. In 2004, the crystal structure of T. emersonii
(or Rasamsonia emersonii) CBH IB (TeCel7A) represented the
first structure of a thermostable GH7 CBH and also the first of a
GH7 CBH naturally lacking a CBM (PDB code 1Q9H).464 In
general, the structure of TeCel7A is quite similar to TrCel7A,
with two exceptions. First, the tip of the B2 loop was not
resolved in the TeCel7A crystal structure, so comparison is not
possible. Also, the A1 loop at the binding tunnel entrance is
extremely similar to that of PcCel7D (Figure 30A), which is
somewhat shortened from that of TrCel7A.464
6.1.10. PcCel7D Bound with Disaccharides. Further
structural studies of PcCel7D in 2005 focused on the interaction
with inhibitors: natural product cellobiose (PDB code 1Z3T),
lactose (PDB code 1Z3V), and cellobioimidazole (PDB code
1Z3W).458 The structural information revealed here was used to
explain the differences in affinities for cellobiose and lactose
between PcCel7D and TrCel7A. Residues on the B3 loop may
provide more possibilities for interaction with both the substrate
and the product in TrCel7A. TrCel7A, however, lacks a
conserved acidic residue (Asp336) that was shown to interact
1346
with a glucopyranose in the +2 subsite of PcCel7D (Figure
30C). The studies also revealed that Tris (buffer molecule) can
bind in the active site, and that both Tris and calcium inihibit
PcCel7D CBH activity.
6.1.11. M. albomyces Cel7B. The structure of another
thermostable GH7 CBH natively lacking a CBM was published
in 2008 from M. albomyces.465 MaCel7B (PDB code 2RFW) was
crystallized with four molecules within one asymmetric unit and
was determined both in apo form and with bound cellobiose,
cellotriose, and cellotetraose. This gave a structural analysis of
altogether 16 different “structure snapshots” of the same enzyme
and the total picture of a surprisingly large conformational
variability of the substrate interaction with the enzyme. The
structure of MaCel7B is also quite similar to TrCel7A. The most
significant difference from TrCel7A is that MaCel7B possesses a
slightly elongated entrance loop A1, which features a unique
tyrosine residue (Tyr100, which neither PcCel7D nor TrCel7A
possess) that sits “above” the −7 binding site, opposite the
conserved tryptophan at this site (Trp40 in TrCel7A).
6.1.12. Heterobasidion irregulare Cel7A. The comparison
of PcCel7D and TrCel7A was taken one step further by Momeni
and Payne et al. in 2013.449 This publication presented the
structure of Cel7A from the root-rot fungus and basidiomycete
H. irregulare (PDB code 2YG1). HirCel7A also possesses the
elongated A1 loop seen in MaCel7B, including the extra tyrosine
residue (Tyr101 in HirCel7A; Figure 30A).465 HirCel7A has a
slightly shorter B3 loop (deletion of two residues) compared
with TrCel7A (and PcCel7D lacks this loop entirely), resulting
in the loss of stable contacts across the binding tunnel with loop
A3 that TrCel7A possesses (Figures 29 and 30B). Structural
comparisons of the three enzymes were complemented by MD
simulations to examine the flexibility of tunnel closing loops and
the potential role for these in the recognition of substrate, endo-
initiation, and the processive action of the enzyme. On this basis,
it was suggested that HirCel7A exhibits intermediate properties
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between TrCel7A and PcCel7D in terms processivity and

possible endo-initiation capability.
6.1.13. T. harzianum Cel7A. In 2013, the structure of T.
harzianum Cel7A (ThCel7A; PDB code 2Y9L), an enzyme with
81% sequence identity with TrCel7A, revealed a few significant
structural differences.466 The entrance to the substrate-binding
tunnel is more open in ThCel7A than TrCel7A, due to the
shortening of the A1 loop, very similar to that seen in PcCel7D
(Figure 30A) and TeCel7A. The second highlighted difference
was the potentially greater flexibility of the B3 loop (denoted
loop 4 in the original publication) as a result of a single residue
substitution between ThCel7A (Ala384) and TrCel7A
(Tyr371). This substitution is not on the B3 loop itself, which
is highly conserved between the enzymes, but on the opposing
side of the tunnel (Figure 30B). MD simulations of the two
enzymes confirmed that this substitution does increase the
flexibility of the B3 loop and results in a more open binding
tunnel.466
6.1.14. L. quadripunctata Cel7B. The year 2013 also
featured publication of the first structure of a nonfungal GH7
CBH from the marine woodborer L. quadripunctata.383 Four
high-resolution LqCel7B structures (PDB codes 4GWA, 4HAP,
4HAQ, and 4IPM) constituted the basis for a structural analysis
and comparisons with TrCel7A using MD simulations. LqCel7B
was shown to have a highly acidic surface charge, which may be
the source of its high activity in saline environments (Figure 31).

Figure 32. Michaelis complex and glycosyl-enzyme intermediate of

TrCel7A. (A) TrCel7A Michaelis complex (PDB code 4C4C441). Note
the 4E conformation of the substrate at the −1 site. (B) TrCel7A
glycosyl-enzyme intermediate (PDB code 4C4D441) with covalent
bond between the nucleophile and the broken cellooligomer chain. The
cellobiose product is in unprimed glycosyl-enzyme intermediate mode.
Note the approximately 30° rotation of the nucleophile during
glycosylation.

Figure 31. Electrostatic potential distribution on the solvent accessible

surface of LqCel7B. Electrostatic potential between −7 kT/e and 7 kT/
e is shown as a colored gradient from red (acidic) to blue (basic). (A)
LqCel7B possesses an anomalously high frequency of acidic residues on
its surface. These are likely required for activity in its native marine
environment. (B) The surface charge of TrCel7A is much less acidic.
Adapted from ref 383.
At the substrate tunnel entrance LqCel7B exhibits the same A1
loop elongation, with extra tyrosine (Tyr121 in this case) also
seen in MaCel7B465 and HirCel7A (Figure 30A).449 The B3
loop conformation for LqCel7B is quite similar to that of
HirCel7A (PDB code 2YG1). HirCel7A and LqCel7B both lack
the tyrosine (Tyr247) that TrCel7A possesses that interacts
with loop A3 across the binding tunnel (Figure 30B). Where
LqCel7B and HirCel7A differ though is in the tyrosine (Tyr371
in TrCel7A) on the opposing A3 loop: LqCel7B possesses this
tyrosine, whereas HirCel7A does not (Figure 30B).
6.1.15. TrCel7A Michaelis Complex and Glycosyl-
Enzyme Intermediate. In 2014, two structural snapshots of
key steps in the GH7 retaining mechanism were published
(Figure 32), namely the Michaelis complex (PDB code 4C4C)
and glycosyl-enzyme intermediate (PDB code 4C4D) of
TrCel7A, both as acid/base disabled mutants (E217Q).441
1347
The Michaelis complex featured a cellononaose chain that spans
the entire binding tunnel. The glycosyl-enzyme intermediate
contains a cellohexaose molecule covalently bound to the
nucleophile and a cellobiose product filling the +1/+2 sites. The
glycosyl-enzyme intermediate was captured by the highly
successful method206,451−453 of incubation with 2,4-dinitro-
phenyl 2-deoxy-2-fluoro-β-cellotrioside, a mechanism-based
suicide inhibitor.170,206,452−454 These structures constitute the
first experimentally determined structural picture of the
Michaelis complex for a GH7 CBH (confirming much of the
geometry in the theoretical model of the Michaelis complex
presented in 1998 with PDB code 8CEL173) and the first
glycosyl-enzyme intermediate for any GH7 enzyme.441
6.1.16. GH7 Catalytic Insights from Molecular Simu-
lation. Connecting the many geometrical changes between the
static configurations captured in crystal structures with the
dynamical variables that drive chemical reactions necessitates
the use of computational modeling and simulation. Moreover,
modeling is essential for the computation of individual free
energy barriers and rates of fundamental process
steps.111,159,467,468 This makes computational studies crucial to
the development of enzymatic structure−function relation-
ships.111 To that end, many computational studies have
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Figure 33. Hydrolytic free energy barriers for TrCel7A. Free energy barriers for the hydrolytic steps of glycosylation (left) and deglycosylation (right)
for TrCel7A acting on a cellulose chain are shown in addition to that for the nucleophilic water movement that is coupled to cellobiose product
movement (middle).441 Rate calculations based on these free energy barriers reveal that glycosylation is rate-limiting within the hydrolytic steps. M
denotes the Michaelis complex.
examined the chemical steps of GH7 enzymes, both of EG
TrCel7B469 and CBH TrCel7A, as reviewed below.441,470−472
Zhang et al. used hybrid QM/MM (quantum mechanics/
molecular mechanics) calculations to compute the two-dimen-
sional potential energy surface as a function of the key breaking
and forming bonds for both hydrolytic steps of p-nitrophenyl-β-
469
D-lactoside (pNPL) hydrolysis by EG TrCel7B. They found
that the barrier to glycosylation (18.9 kcal/mol) is higher than
the barrier to deglycosylation (10.5 kcal/mol). Site-directed
mutagenesis experiments were also performed, with the goal of
attributing functional roles for several near-active site residues
that are well-conserved in GH7 enzymes. R108 K, Y146F,
Y170F, and D172N mutants revealed catalytic activities that
were decreased between 130- and 7700-fold. Subsequent MD
simulations of these mutants revealed a disrupted hydrogen
bond network that resulted in more distant interactions with the
catalytic residues, thus hindering either the nucleophilic attack
or the proton transfer. This likely increases the catalytic free
energy barriers, explaining the observed reduction in activity and
underscoring the catalytic importance of the environment near
the active site beyond simply the “catalytic residues”.
Li et al. utilized QM and QM/MM to perform single-point
calculations for both steps of the hydrolytic mechanism of
TrCel7A.471 Their QM calculations indicate that the free energy
barrier for step 2 is more than twice as high as that for step 1 at
more than 30 kcal/mol (compared to a glycosylation barrier of
around 14 kcal/mol). Yan et al. performed QM/MM umbrella
sampling on wild-type TrCel7A as well as E212Q and D214N
mutants.472 They only study step 1 of the hydrolytic mechanism
finding a free energy barrier of more than 30 kcal/mol in all
three cases. In addition to being difficult to reconcile with one
another, these free energy barriers are difficult to reconcile with
experimental hydrolysis rates which would suggest a significantly
lower hydrolytic barrier.
Barnett et al. also utilized QM/MM umbrella sampling
simulations for step 1 with wild-type TrCel7A,470 utilizing two
coordinates (one breaking and one forming bond) along which
to sample free energy. Their prediction of the step 1 free energy
barrier was 17.5 kcal/mol, from which transition state theory
predicts a reaction rate of 0.4 s−1. This study also presented
details on the ring puckering itinerary for step 1 and the
importance of the oxocarbenium character of the transition
state, as described in section 3 for GH catalysis.
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Review
Knott et al. calculated free energy barriers for both steps 1 and
2 for TrCel7A.441 Path sampling simulations were utilized to
elucidate the reaction mechanism prior to computing free
energies.473−475 This is in contrast to previous computational
investigations in which free energies were computed along
assumed and unverified coordinates (e.g., bond lengths). Path
sampling revealed that the glycosylation reaction coordinate
(Figure 33, “RC1”) contains components of the forming and
breaking bonds as well as a rotation in the nucleophile. This
finding was corroborated by the crystallographic snapshots
presented in the same study of the Michaelis complex (PDB
code 4C4C) and glycosyl-enzyme intermediate (PDB code
4C4D). Comparing the conformation of nucleophile Glu212 in
the two structures (Figure 32) reveals an approximately 30°
twist, consistent with the rotation seen in the simulations. In
between the two hydrolytic steps, the cellobiose product cleaved
during glycosylation shifts slightly toward the tunnel exit in
order to allow the nucleophilic water access to the anomeric
carbon reaction center. The free energy barrier for this transition
was computed along a distance coordinate describing the
proximity of this water to the active site (Figure 33). An
additional noteworthy aspect of this work was the finding that
deglycosylation proceeds via a product-assisted mechanism: the
reaction coordinate (Figure 33, “RC2”) involves forming and
breaking bonds as well as the orientation of a hydroxyl from the
cellobiose product, which positions the catalytic water for
nucleophilic attack on the anomeric carbon. Subsequent free
energy and dynamic calculations facilitated TST rate calcu-
lations of 10.8 and 5300 s−1, for steps 1 and 2, respectively, thus
confirming step 1 as rate-limiting in the hydrolytic cycle. This
rate agrees well with the rate of processive cellulose hydrolysis
by TrCel7A on crystalline cellulose measured by Igarashi et al.
via high-speed AFM of 7.1 ± 3.9 s−1.476
In addition to these studies that explicitly studied bond-
breaking and bond-forming in GH7 enzymes, several other
computational studies have helped to shed light on other factors
influencing enzymatic catalysis. These factors include substrate
ring distortion,477 protonation of catalytic residues,478,479
mutations,472,480 and protein interfacial allostery.481,482 In
addition, the glycosynthetic ability of the nucleophilic mutant
(HiCel7B E197S mutant) was examined via QM/MM
metadynamics simulations.480
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Figure 34. Complete processive cycle of a GH7 CBH. TrCel7A is shown with its CD, linker, and CBM in gray “cartoon” representation. N-
glycosylation and O-glycosylation are shown in blue and yellow, respectively. The cellulose surface is shown in green and the cellobiose product in
magenta. Following the adsorption of the CBM and CD to the substrate and initial chain threading, TrCel7A processively cleaves cellobiose from a
cellulose chain end. The “Processive Cycle” includes chain processivity, hydrolysis, and product expulsion (Figure 35). This processive cycle occurs
repeatedly until the enzyme desorbs from the cellulose surface.

Figure 35. Hypothesized hydrolytic processive cycle of a GH7 CBH inferred from structural data. The inner processive cycle of a GH7 CBH consists
of the following seven steps: after adsorption, decrystallization, and initial chain threading, the substrate fills the −7 to −1 sites in pre-slide mode
(upper left); (1) processive motion of CBH by one cellobiose unit fills the product sites, with all glucosyl residues in the stable chair conformation
(slide mode, upper middle); (2) catalytic activation rotates the chain ∼90° and distorts the −1 glucosyl residue into half-chair or envelope
conformation, forming the Michaelis complex (upper right) and allowing the nucleophile access to the anomeric carbon reaction center; (3) the first
chemical step, glycosylation, cleaves cellobiose from the reducing end of the cellulose chain and forms a covalent bond between the nucleophile and
the broken chain (unprimed glycosyl-enzyme intermediate, lower right); (4) a shift in the product produces the primed glycosyl-enzyme intermediate
(lower middle) such that the deglycosylation nucleophilic water has access to the anomeric carbon reaction center; (5) the second chemical step,
deglycosylation, produces the product complex, wherein the glycosyl-enzyme intermediate is broken and the catalytic residues are regenerated (lower
left); (6) product expulsion vacates the +1/+2 sites (upper left). The processive cycle ends when the enzyme dissociates from the cellulose chain. It
should also be noted that it is possible for a CBH to perform endo-type initiation,304,307 in which case the enzyme would initiate this cycle in slide
mode (or similar, possibly with a longer chain on the product side of the active site) and the remainder of the processive cycle would proceed as
depicted. In all panels, the nucleophile (Glu212 in TrCel7A) is on top, and the catalytic acid/base (Glu217 in TrCel7A) is on bottom in green “sticks”.
For clarity, only selected hydrogen atoms are shown: the hydrogen bonded to the −1 anomeric carbon shows the stereochemistry and that of Glu217
shows its protonation state throughout the processive cycle.

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6.2. Processivity, Kinetic Modeling, and Visualization
The wealth of structural and biochemical data on GH7 enzymes
has revealed a common overall fold as well as a common
catalytic machinery. Clues have also been provided by structures
as to differences in functionality (e.g., processivity) between
CBHs and EGs and well as within these classes. This section
discusses the discrete steps in the processive cycle of GH7
enzymes, with a particular focus on CBHs. Visualization studies
of GH7 enzymes on lignocellulosic biomass are then reviewed,
as they predominantly provide qualitative information about
cellulase action. Kinetic modeling and novel high-speed AFM
measurements make this knowledge quantitative and have the
capability to provide rates for the individual steps of the
processive cycle. We then discuss structural and molecular
modeling studies, which allow for identification of the molecular
underpinnings of cellulase action including the individual steps
of the processive cycle. Finally, the reversible blockage of this
processive cycle via product inhibition is discussed and
reviewed.
GH7 CBHs act from the reducing end of cellulose chains445
and perform many hydrolytic events before disassociating from
a cellulose chain. This overall processive cycle includes at least
the following steps (Figure 34): adsorption to the crystalline
cellulose surface (possibly preceded by the adsorption of an
attached CBM), cellulose chain decrystallization, chain thread-
ing through the binding tunnel or direct binding (i.e., endo-
initiation304), hydrolysis, product expulsion, and desorption.
The “repeating processive” cycle that generally repeats many
times before chain dissociation includes processive motion,
catalytic activation, hydrolysis, and product expulsion (Figure
35). The rate-limiting step in the processive cycle of a cellulase is
an oft-discussed topic in the literature, due to its importance as
the primary target for protein engineering efforts.
For an EG, the processive cycle shown in Figure 35 is
modified in that the chain threading and product expulsion are
omitted. Chain acquisition without threading is thought to be
readily accomplished by EGs (as opposed to CBHs) due to their
open binding site clefts that lack the enclosing loops found in
CBHs. GH7 CBHs are also capable of performing hydrolysis in
an endo-fashion.304,307 It has been speculated that, with the
ability for CBHs to perform endo-initiation, their enclosing
loops may open, allowing entry of the cellulose chain into the
active site without chain threading.304,307 Whether or not a CBH
performing endo-initiation subsequently disassociates immedi-
ately or embarks on a processive run is still an open question.
Quantifying the processive ability of an enzyme is useful both
in generally describing the mechanistic behavior and in
identifying opportunities for activity enhancements. Unfortu-
nately, measuring processivity is not straightforward, and
measurements performed via different techniques are not
readily comparable.521 Formally, processive ability is defined
as the number of hydrolytic events performed per number of
initiated processive runs. This definition of processive ability is
frequently referred to in the literature as “apparent processivity”.
Kurašin and Väljamäe describe a complementary measurement,
“intrinsic processivity”, that describes the theoretical processive
potential of a GH, in the limit of ideal polymeric substrate
turnover.307 A handful of techniques have been developed to
assess apparent and intrinsic processivity, some of which are
described below. These methods generally capitalize on the
relatively consistent nature of the processive GH product
profile.307,522−531 However, characterization of processive ability
by measuring produced soluble products requires some
1350
Review
assumptions be made regarding initial binding mode (i.e.,
endo vs exo, where some processive enzymes are known to
exhibit both initiation modes) and can frequently lead to
misinterpretation or overestimation of processivity.461,532
Furthermore, these methods are also extremely sensitive to
the substrates selected, where the number of available free chain
ends can drastically affect measured product profiles.533 A recent
review provides an excellent assessment of the pros and cons of
each available technique.521 Finally, we caution the reader to
approach experimental determinations of processivity with an
eye toward the difficulty in quantification and exercise good
judgment with respect to interpretations of processive ability
and claims of endo- or exo-initiated binding.
The direct in situ visualization of cellulase action on cellulosic
substrates has provided valuable clues to their mechanistic
action and has recently been reviewed.534,535 These visualization
methods offer the ability to resolve single molecules on the
cellulose surface, allow for observing single cellulases interacting
with the cellulose surface, and provide temporal tracking of the
effects of cellulases on the cellulose surface (e.g., the formation
of fissures). TEM was the first method applied to visualizing the
structural dynamics of enzymatic cellulose degradation directly
on the cellulose surface both for the complete T. reesei system536
and for TrCel7A in isolation.537
White and Brown visually presented the EG/CBH synergistic
action of T. reesei enzymes.536 They found that CBH or EG in
isolation could not produce cellulose microfibril degradation,
though EG could produce some splaying of chains (notably,
results were only reported for 1 h of incubation). When acting in
concert, however, they could completely dissolve microfibrils
within 30 min of incubation.
Particularly notable among these early investigations were
those of Chanzy and co-workers on GH7 CBHs.537,538 These
studies provided direct visual information regarding processive
CBH action and have had a long-lasting impact on the field.
Early work showed the binding of TrCel7A to crystalline
cellulose and its subsequent degradation. Significantly, complete
degradation of crystalline cellulose was shown by TrCel7A in
isolation (within 48 h), without the need for any EG present,
producing cellobiose as the major product (determined by
HPLC).537 A follow-up study visualized gold nanoparticle-
labeled TrCel7A molecules via TEM. TrCel7A retains 60% of
the hydrolytic activity even with the bulky gold label (5 nm in
diameter, the same order of magnitude as TrCel7A). It was
found that TrCel7A binds preferentially to the hydrophobic
(100) face of cellulose microfibrils.538
Several other TEM studies focused on GH7 enzymes,536−544
including those utilizing gold labeling538,539 and labeling with
gold-coupled monoclonal antibodies (immuno-EM).541,544
Immuno-EM was utilized to determine that TrCel7A and
TrCel7B preferentially bind to the crystalline and amorphous
regions of substrate, respectively.544
One limitation of TEM is that it cannot visualize hydrated
cellulases; thus, it cannot be used in the native cellulose/
cellulase environment. Conventional atomic force microscopy
(AFM), however, can be applied in liquid environments at
atmospheric temperature and pressure, without sample
modification (labeling, coating, etc.). Thus, AFM enabled the
first visualization of cellulase action in a biologically relevant
environment.545 Subsequent AFM studies revealed further
details of enzymatic action.367,545−550 For example, AFM
visualization of TrCel7A action on crystalline cellulose
suggested, under environmental conditions, that degradation was
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primarily on a single face.367 When combined with the previous
finding from TEM that the family 1 CBM of TrCel7A
preferentially binds to the hydrophobic face of crystalline
cellulose (as described in section 5),362 this may indicate
TrCel7A localization primarily to the hydrophobic face of
crystalline cellulose. This confirmed in an aqueous environment
and with the native enzyme what Chanzy et al. had previously
found via TEM of gold-labeled TrCel7A.538 AFM also showed
the formation of path-like indentations on the substrate when
incubated with TrCel7A, considerably different than the effect of
EG,547 and that the processive motion of TrCel7a is impeded by
amorphous regions of the substrate.546 In addition, AFM has
been utilized to study synergistic effects between TrCel7A with
TrCel6A and EGs546,551,552 and in HiCel7A with Cel6A540 as
well as with EG present.553 Real-time AFM has also examined
EG action by itself (TrCel7B).549 Fluorescently labeling
TrCel7A coupled with confocal microscopy554 or total internal
reflection fluorescence microscopy555 allows for tracking the
CBH’s motion along cellulose fibrils. The latter of these studies
found that upward of 90% of TrCel7A molecules were stationary
on the cellulose surface during the observation interval.555
These studies highlight the utility of visualization studies in
enhancing the understanding of cellulase−cellulose dynamical
interactions as well as cellulase−cellulase interactions in some
cases.
The general insights provided by cellulase visualization have
been solidified, refined, and quantified by kinetic modeling
studies and novel HS-AFM measurements. These studies
provide the capability of determining rate constants for the
individual steps in the processive cycle (Figures 34 and 35) and
how these constants depend on the nature and concentration of
both substrate and enzyme. These models are often directly
linked to hydrolysis experiments providing feedback between
kinetic experiments and theories that seek to rationalize the
results. The goals of these kinetic models are often focused on
identifying the rate-limiting step(s) in the processive cycle of a
CBH or an enzyme cocktail. Cellulase synergy and the origin of
the “burst phase” often seen in the first few minutes of
hydrolysis experiments are two other primary areas of focus of
these studies. With some notable exceptions, we largely focus
our attention here on those studies that have appeared from
2009 to the present, as both Zhang and Lynd33 and
subsequently Bansal et al.556 have nicely reviewed the literature
on this topic for publications appearing before 2009.
Ståhlberg et al.348 examined the adsorption and hydrolysis of
intact TrCel7A, isolated CBM, and isolated catalytic core on
MCC. They found that the CBM increased both the adsorption
and hydrolytic activity. To explain these results, they proposed a
model for the action of two-domain cellulases: the core is biased
toward amorphous regions or chain ends whereas the CBM is
biased toward the crystalline regions. Attachment of the CBM to
the core ensures that the core will have an elevated
concentration on the crystalline regions, thus increasing the
probability of cleavage. The importance of considering at least
two different types of surface morphologies was thus
emphasized.
Beginning around 2009, multiple studies were presented that
revolutionized our quantitative understanding of CBH
action.307,315,476,531,557−562 Taken together, they constitute a
significant step forward in our collective understanding of CBH
function, beginning with the seminal study from Igarashi et al. in
which high-speed AFM was utilized to spatially track individual
TrCel7A molecules on crystalline cellulose with a temporal
1351
Review
resolution of 1−4 frames/s.558 Isolated CDs (sans CBM) moved
with comparable velocity to intact enzymes with CBM at around
3.5 nm/s. However, E212Q (catalytic nucleophile) and W40A
(binding tunnel entrance) mutants do not move at all. Thus, it
was concluded that hydrolysis and chain loading are both critical
for movement. The E212Q mutant was immobilized on the
surface longer than the W40A mutant, suggesting W40 is critical
for initial chain threading. On the basis of the observations
regarding the immobile E212Q mutant and the comparable
velocities of intact enzyme and isolated CD, they concluded that
movement is inherently coupled to catalytic activity. While
possible that the cleavage reaction actually induces the forward
processive motion, these observations at least imply that the
catalytic event is a prerequisite for motion to continue.
Additionally, the role of the CBM was determined to be solely
to enhance the concentration of enzyme molecules on the
substrate, without a further substrate-modifying role.
In 2010, Jalak and Väljamäe sought to identify the cause of the
rapid rate reduction characteristic of enzymatic hydrolysis531 by
quantifying the concentration of CBHs with a cellulose chain
productively bound to cellulose (meaning that the cellulose
chain was bound in the active site). Experimentally, this was
accomplished by measuring the degree of inhibition of TrCel7A
and PcCel7D for a small MW reporter molecule, in this case
pNPL. The rate of pNP production (product of pNPL
hydrolysis) is readily related to the fraction of CBH with free
active site (and thus the productively bound fraction). The
observed catalytic constant can be calculated as the ratio of
cellobiose formation rate (cellulose hydrolysis product) to the
concentration of CBHs with occupied active site. As candidates
for the source of the rapid rate retardation, previously proposed
hypotheses such as cellobiose product inhibition, depletion of
more readily hydrolyzable substrate, depletion of available chain
ends, inactivation through irreversible surface binding, and
overcrowding of bound CBHs could not account for their data.
However, if “steric obstacles” that limit the processive action of
CBHs were considered, the results could be rationalized. In this
way, the enzyme dissociation rate from the surface (koff) is
implicated as the slow step of the overall CBH processive cycle
with only CBHs present. Following the initial “burst” phase, the
rate of hydrolysis is governed by koff and the average obstacle-
free path of a CBH on a cellulose chain. This explanation also
accounts for the differences in hydrolysis rates wherein CBHs
with more open binding tunnels (e.g., PcCel7D vs TrCel7A)
more readily dissociate from cellulose chains, resulting in lower
processivity, but higher cellobiose production. Thus, there are
“costs and benefits” to high processivity.563,564 The concept of
steric obstacles that limit CBH catalytic production has been
quite influential in subsequent studies performed by a number of
groups.
The concept of a CBH processing along a cellulose chain
according to its “true” catalytic constant before becoming
“stuck” at an obstacle necessitates (at least) two major CBH
populations. In 2011, Igarashi et al.476 presented high speed
AFM data of TrCel7A as well as TrCel6A that provided a
powerful visual verification of this “dual population” model. In
this study, TrCel7A was observed to alternate between stopping
and sliding. The velocity distribution of CBHs could be very
well accounted for by assuming two populations, one with near
zero velocity and the other with nonzero velocity (7.1 ± 3.9 nm/
s). CBHs were shown to stop, and then accumulate behind a
surface obstacle (described as a “traffic jam”). A new dimension
was added to this dynamic picture when a group of CBHs was
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Figure 36. Cellulase architecture correlates with function. Shortened or absent substrate-enclosing loops result in a more open architecture which
gives rise to functional differences. The rate constants and processivity estimates are from Kurašin and Väljamäe.307 (A) CBH TrCel7A has the most
closed substrate-binding tunnel (PDB code 1CEL172), which gives rise to the highest Pintr and lowest koff of the enzymes considered in the study. The
ligand shown is from the TrCel7A Michaelis complex (PDB code 4C4C441). (B) CBH PcCel7D (PDB code 1GPI460) has a more open substrate-
binding tunnel than TrCel7A due mostly to the shortening of the A1 and B3 loops (Figure 29). The ligand shown is from the TrCel7A Michaelis
complex. (C) EG TrCel5A (PDB code 3QR3566), shown in complex with the ligand from Bacillus agaradhaerens Cel5A (PDB code 4A3H206). (D) EG
TrCel12A (PDB code 1H8V567) shown with the ligand from Humicola grisea Cel12A (PDB code 1UU6208). In all panels, the enzymes are shown to
scale and oriented with the acid/base on top and the nucleophile on the bottom (all four utilize a retaining mechanism). The substrate for each of the
307
listed measurements is amorphous cellulose, with the exception of PendoBC , which was performed on BMCC.

seen to stop, accumulate, and then resume motion without
dissociating. This may correspond to the collective action of
several CBHs removing an obstacle in their path. TrCel6A by
itself was observed to bind, but it did not slide nor did it
appreciably degrade cellulose. The combination of TrCel6A with
TrCel7A degraded cellulose much more efficiently than the sum
of the action of the two, giving a powerful visual representation
of CBH synergy, and it was suggested that this may be due to
TrCel6A making endo-like cuts in the crystalline cellulose
surface.
Kurašin and Väljamäe built upon this quantification of the
overall processive cycle by isolating its individual steps and
connecting cellulase functional properties to structure.307 The
resulting seminal study provided an unprecedented holistic
understanding of CBH action in isolation. Recognizing both the
importance of cellulase processivity and also the difficulty in its
measurement, Kurašin and Väljamäe sought to develop a robust
method for its quantification. They note that the intrinsic
processivity Pintr (determined by the ratio of the hydrolytic rate
kcat to the dissociation rate koff) can only be realized on a
“perfect” polymer. On real substrates, the inherent hetero-
geneity introduces steric obstacles that halt forward processive
motion. Thus, they sought to measure the ratio of the number of
actual catalytic events before dissociation on a real polymer (i.e.,
the apparent processivity Papp). The primary experimental
challenge for the quantification of apparent processivity is
determination of the number of processive run initiations. This
was elegantly addressed by selectively “tagging” only the
reducing ends of cellulose with diaminopyridine (DAP) and
detecting the release of DAP-labeled sugars (after cleavage by
reducing end specific CBHs TrCel7A and PcCel7D) with
sensitive fluorescence detection. This analysis revealed that Papp
for the CBHs increased with increased enzyme loading,
indicating that there was another mode of initiation besides
exo-action, namely endo-type initiations. This led them to
perform similar experiments with reduced BC to measure the
number of endo-initiation events. Endoinitiation capability by
1352
TrCel7A had previously been reported,304 but there was some
uncertainty about these findings.565 TrCel7A was shown to
perform endo-initiation with probability Pendo of 0.41−0.55 on
BC (PcCel7D, Pendo = 0.73−0.82) and 0.83−0.93 on amorphous
cellulose (PcCel7D, Pendo = 0.92). Also, the Papp of TrCel7A was
similar to that of PcCel7D on amorphous cellulose and on BC;
however, Papp for each CBH was nearly 3 times higher on BC,
leading to the conclusion that this parameter is determined by
the substrate properties. The dissociation rate koff was also found
to be dependent on the nature of the substrate. Moreover, the
koff values for the EGs were 2 orders of magnitude above those
of the CBHs. This is in contrast to kcat, which was of the same
order of magnitude for the CBHs as for two EGs (TrCel5A and
TrCel12A) and for the different types of cellulose. Pintr was also
determined by separately estimating kcat and koff. These rate
constants were determined by measuring product formation rate
in the regimes where the processes described by these constants
are rate-limiting (the initial “burst” phase for kcat and the
subsequent linear regime for koff). Pintr was 1−2 orders of
magnitude greater than Papp, confirming that CBHs do not reach
their full processive potential on real substrates, and thus
processivity is substrate limited. The authors thus conclude that
the dissociation of a stalled, nonproductively bound CBH is the
rate-limiting step in the overall processive cycle and the primary
target for the selection of cellulases, though it must be noted that
this conclusion is based on measurements of individual enzymes
in isolation (e.g., a CBH in the absence of any synergism with an
EG, other CBH, or LPMO). The trends in various measured
parameters (koff, Pendo, Pintr) are indicative of a deep connection
between enzyme structure and intrinsic kinetic properties
(Figure 36). The key structural difference between EGs and
CBHs is the shortening or deletion of several loops in EGs that
cover the substrate binding tunnel (Figures 25, 27, and 28).
Also, PcCel7D has a shortening of the important B3 loop (aka
the “exo” loop) as compared to TrCel7A (Figure 29). These
loop differences are the likely structural basis for the increased
Pendo of PcCel7D versus TrCel7A (and even higher Pendo for
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Figure 37. CBHs acting in isolation and the two modes of endo/exo synergism. (A) The rate of cellobiose production by CBHs acting in isolation (i.e.,
no EGs present) is limited by dissociation from cellulose when hindered by obstacles (most notably the amorphous regions of cellulose).307 (B) The
traditional picture of endo/exo synergism (right side of panel B) is that EGs create random cuts in the cellulose surface that provide starting points for
CBH processive action. Jalak et al.557 showed that another important role for EGs was to help CBHs “escape” blockage (left side of panel B) by
amorphous regions of cellulose, as shown in panel A. Reprinted with permission from ref 557. Copyright 2012 the American Society for Biochemistry
and Molecular Biology, Inc.

EGs). In addition, PcCel7D more easily dissociates from a
cellulose chain than TrCel7A (reflected in a higher koff, and thus
a lower Pintr). The fact that the koff for the EGs studied is 2 orders
of magnitude higher than for the CBHs also seems to implicate
loop structures as the source of the differences in activity of
these cellulases (Figure 36).
Similarly, Praestgaard et al.562 developed an explicit kinetic
model to describe the “burst” kinetics seen in cellobiose
production by CBHs, such as Cel7A wherein the steady-state is
preceded by a transient burst in activity. They compare their
results to calorimetric measurements performed with TrCel7A.
The key components of the theory are the relative reaction rate
constants for adsorption, processive hydrolysis, and desorption,
as well as blockage by obstacles on the cellulose surface. They
find that the burst in activity persists until the CBHs start
encountering obstacles in significant proportion. Their model is
capable of capturing this burst phase; however, to capture the
double exponential decay in cellobiose production rate seen
experimentally, it was necessary to add random enzyme
inactivation (with no correlation to the stage of the processive
cycle) to their model. This model was further developed and
applied to further mine the experimental wealth of mechanistic
information on fast (i.e., non-rate-limiting) steps of the
processive cycle afforded by the pre-steady-state regime.560
This constituted the first such pre-steady-state investigation of a
cellulase on its natural substrate, insoluble cellulose. For
TrCel7A, it was found that the rate of cellobiose production
reaches a maximum after just 5−8 s, and then declines rapidly.
They calculate 4 glycosidic bonds cleaved per second (kcat),
concluding that dissociation (koff) is rate-limiting (0.022/s).
Complexation (kon) is 3× faster than dissociation (koff), and kcat
(which includes hydrolysis, processive motion, and product
expulsion) is 2 orders of magnitude faster than either kon or koff.
The stage was now set for a paradigm shift in the
understanding of cellulase synergism. Although some studies
1353
had indicated a role for EGs in “surface cleaning”,568,569 the
prevailing model of endo/exo synergism posited that EGs make
internal cellulose chain cuts that produce starting points for
CBH action.570 However, the recent revelation that the rate-
limiting step for processive CBH action in isolation was
dissociation (Figure 37A) and not association,307 suggested that
the synergistic power of EGs might somehow be related to CBH
dissociation. Jalak et al.557 demonstrated that the hydrolytic rate
constant of TrCel7A on BC increased with the addition of EG
TrCel5A under steady-state conditions, unaccountable by the
traditional paradigm. This led to the new hypothesis: the role of
EGs is not only to help CBHs attach to the cellulose surface, but
also to detach f rom the cellulose surface (Figure 37B), the step
which was now gaining traction as rate-limiting in the CBH
processive cycle (though EGs themselves were prone to
reversible deactivation by surface heterogeneity315). Both
modes of synergy were found to act in concert, but the
traditional paradigm accounts for a significant portion of the
synergistic effect only at high enzyme/substrate ratios.557 At
optimal enzyme/substrate ratios, the new synergistic mecha-
nism dominates. Under these conditions (optimal enzyme/
substrate loadings and with EG present), the result is that the
rate-limiting step for the conversion of cellulose to glucose is the
CBH processive cycle.557 In other words, the combined steps of
processive motion, hydrolysis, and product expulsion (Figure
35) are rate-limiting in the overall processive cycle which
includes association and dissociation (Figure 34). This proposal
was corroborated by the fact that, at these optimal enzyme/
substrate loadings, kobs (calculated from the rate of cellobiose
production normalized by the number of TrCel7A molecules
with occupied active site) approached kcat (the rate constant for
the processive cycle involving hydrolysis, product expulsion, and
processive motion, Table 6). These findings were also
demonstrated to be true on lignocellulose.557 In addition, it
was noted that the apparent processivity calculated for TrCel7A
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Figure 38. Free energy landscape of the hydrolytic and processive steps for TrCel7A. The barriers for glycosylation,441 product movement,441
deglycosylation,441 processive motion,459 and catalytic activation459 were computed via advanced molecular simulation techniques. These barriers
represent all of the key steps in the CBH processive cycle (in between adsorption and desorption) with the exception of product expulsion, which has
previously been experimentally ruled out as a rate-limiting factor in these enzymes.557 Because product expulsion is not explicitly considered here, the
overall free energy change for the processive cycle will not be as dramatic as shown. Taken with previous results, these findings led to the conclusion
that the glycosylation reaction is the rate-limiting step in the enzymatic deconstruction of cellulose by TrCel7A.459

is similar to the DP of the BC substrate (DP ∼ the length of
obstacle-free path = nfree); thus, the “obstacles” were identified
with the amorphous regions of the BC (Papp ∼ DP ∼ nfree),
suggesting that CBH is not able to digest in the amorphous
regions.557 When a CBH encounters an amorphous region of
substrate, it stalls and must dissociate before it can continue
hydrolyzing the substrate. Endolytic cuts made in the substrate
provide “escape routes” for the CBH before it reaches the
amorphous regions and facilitate avoidance of the amorphous
regions. An extremely relevant extension of this work would be
to perform similar experiments with other cellulolytic enzymes
including nonreducing end specific CBHs, EGs from other GH
families, and/or LPMOs to understand their molecular-level
synergistic effects.
The rate-limiting step of the processive cycle of a CBH had
now been narrowed to those steps that comprise the “inner
processive cycle” (Figure 35), namely, processive motion,
catalytic activation, glycosylation, product movement, deglyco-
sylation, and product expulsion.557 These are also the steps that
take place in the observed CBH processive motion in high-speed
AFM studies.476,558,564 Further narrowing which of these steps
limits the overall production of cellobiose would be extremely
difficult to accomplish experimentally, but molecular simulation
has provided insight in this regard. Product expulsion has been
studied via molecular simulation571,572 and has been ruled out as
the rate-limiting step in CBH action.557 The free energy barriers
for glycosylation, product movement, and deglycosylation were
calculated as 15.5, 2.0, and 11.6 kcal/mol, respectively;441 the
reaction rate for glycosylation was found to be much lower than
that for deglycosylation making it the rate-limiting hydrolytic
step (discussed in more detail in section 6.5). Free energy
profiles for the remaining steps of the TrCel7A processive cycle
1354
were elucidated in 2014.459 The barrier for processive motion
(advancing along the cellulose chain by one cellobiose unit) was
calculated via umbrella sampling and found to be higher than
that for catalytic activation (the formation of the Michaelis
complex) at 4.2 and 2.9 kcal/mol, respectively. Both of these
barriers are significantly lower than the 15.5 kcal/mol barrier for
glycosylation (Figure 38).441 The barrier to processive motion
may seem surprisingly small, given the many enzyme−substrate
interactions that must be broken for the cellulose chain to
advance. However, many new and strong interactions are
formed upon chain advancement, and given the flexibility of the
residues lining the binding tunnel, these can start to be formed
before prior interactions are fully broken. Knott et al. pointed in
particular to the strong binding of polar residues at the product
sites that are empty following product expulsion (and
immediately prior to processive motion).459 Taken with
previous computational and experimental results, this computa-
tional evidence suggests that the rate-limiting step in the overall
conversion of cellulose is the glycosylation reaction. As such,
accelerating this step is thus predicted to constitute a primary
target for the engineering of improved GH7 CBHs. In addition,
a significant stabilization of −8.1 kcal/mol results from the
processive motion. This “driving force” for processive motion
was explained in terms of the particularly strong binding of the
leading glucosyl residue of the cellulose chain, which advances to
the +2 binding site. The primary residues responsible for this
coordination (Asp259 and Arg394) are conserved in GH7
CBHs, but not in GH7 EGs (Figure 28). Further analysis of
these simulations also suggested that the aromatic residues that
line the binding tunnel are primarily for providing the tunnel
shape, guiding the cellulose chain to the active site in the correct
orientation and conformation.
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Figure 39. Product binding modes in GH7 enzymes. The various binding poses of the glucosyl residues filling the product sites represented by
TrCel7A crystal structures. These include slide mode (exhibited by PDB code 5CEL),446 cut mode also known as the Michaelis complex (PDB code
4C4C),441 unprimed glycosyl-enzyme intermediate (PDB code 4C4D),441 and primed glycosyl-enzyme intermediate mode (PDB code 3CEL).446 For
reference, the catalytic residues of the unprimed glycosyl-enzyme intermediate (PDB code 4C4D) are shown.
An important theoretical advance in the treatment of
enzymatic adsorption was the utilization of the quasi-steady-
state assumption to give physical significance to the parameters
of the oft-used (and inherently nonprocessive) Michaelis−
Menten (MM) framework.559 The MM framework is often
applied to processive enzymes (e.g., cellulases), because it is able
to fit the experimental data reasonably well. However, the
justification for applying the MM framework to processive
systems had never been firmly established; thus, the fitting
parameters obtained lacked physical meaning. Cruys-Bagger et
al. applied the quasi-steady-state assumption to a set of
processive enzyme reactions, namely, association (kon),
processive hydrolysis (kcat, which includes processivity, hydrol-
ysis, and product expulsion), and dissociation (koff). Thus, they
connected MM parameters with rate constants for the discrete
processive steps of a CBH. Relatively straightforward data
obtained from standard assay techniques could now be
converted to rate constants for the elementary steps via simple
mathematical expressions. This approach was subsequently
applied to the hydrolysis of three different types of cellulose
(Regenerated AC, Avicel, and BMCC) by two variants of
TrCel7A (intact with linker/CBM and cleaved catalytic core),
finding that the on-rate varies with substrate load but not with
enzyme load.561 Dissociation was found to be rate-limiting
except at very low substrate loads, where association became
slower. The CBM was shown to increase the rate on crystalline
but not amorphous cellulose; in fact, cleaving the CBM actually
increased the rate of cellobiose production on RAC. This may
indicate a role for the CBM in substrate disruption (see section
5) or simply indicate a low affinity of the CBM for more
amorphous regions of substrate. This study also found that
dissociation was unaffected by the presence of the CBM
indicating that the dissociation of a cellulose strand from the CD
is slower than CBM disassociation.
Structural and molecular modeling studies have provided
valuable insight into the molecular basis of the processive cycle
of GH7 CBHs. For example, the binding of various substrates
and products in GH7 CBHs provides snapshots of the discrete
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steps in the hydrolytic processive cycle shown in Figure 39.573
Ubhayasekera et al. studied binding in the product subsites for
PcCel7D and TrCel7A, which showed two distinct binding
modes for glucosyl residues in the product sites, referred to as
“cut” and “slide” modes.458 Slide mode was named on the basis
of the proposal that this represents how the product sites are
filled when the cellulose chain first slides across the active site.
Structurally, the TrCel7A structure with two cellotetraose
molecules bound (PDB code 5CEL173) best represents this
stage of the processive cycle. The formation of the Michaelis
complex would then involve a transition (“catalytic activation”)
to cut mode, so-called because it was suggested to represent the
positioning of a glucopyranose immediately prior to enzymatic
cleavage. The recently solved structure of the TrCel7A
Michaelis complex (PDB code 4C4C441) represents this stage.
More recently, these product modes have been refined and
expanded to include the “unprimed glycosyl-enzyme inter-
mediate” and “primed glycosyl-enzyme intermediate” immedi-
ately after glycosylation and before deglycosylation, respec-
tively.573 The unprimed glycosyl-enzyme intermediate mode
essentially overlays cut mode in the product sites and represents
the product position immediately following the first chemical
step (glycosylation). The TrCel7A glycosyl-enzyme intermedi-
ate structure (PDB code 4C4D441) features a covalently bound
cellohexaose molecule in the −6 to −1 subsites and a cellobiose
product in the +1/+2 sites in unprimed glycosyl-enzyme
intermediate mode. While the product remains in unprimed
glycosyl-enzyme intermediate mode, there is insufficient space
between cellobiose and the anomeric carbon reaction center for
the nucleophilic water molecule to approach. Thus, before the
second step of the catalytic cycle (deglycosylation) can proceed,
the product shifts slightly toward the tunnel exit, into the
“primed glycosyl-enzyme intermediate” mode (Figure 35); this
is the position the cellobiose product occupies during the
deglycosylation (exhibited by product complex, PDB code
3CEL446). The “priming” refers to being primed for the second
catalytic step.
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From the first GH7 CBH and EG structures, aromatic
residues that line the substrate-binding tunnel have been
identified as being ubiquitously conserved in GH7 en-
zymes.172,450 The first structure of TrCel7A revealed the
presence of four tryptophan residues lining the binding
tunnel,172 Trp40, Trp38, Trp367, and Trp376, which stack
with the −7, −4, −2, and +1 glucosyl moieties, respectively.
TrCel7B maintains these interactions with one variation: Trp38
in Cel7A is Tyr38 in Cel7B.450 Later crystal structures of
TrCel7A with long oligosaccharides confirmed that these
residues indeed stack face-to-face with the glycosyl moieties of
the substrate.173
Mutation of the aromatic residues in CBH tunnels is typically
significantly detrimental to enzyme activity. In particular,
aromatic residues at the entrance to the binding tunnel have
been show to have a dramatic impact on catalytic function.
Koivula published the seminal study on this topic in 1996.574
Therein, mutation of the leading tryptophan residue in +4
subsite of TrCel6A was demonstrated to abolish activity on
crystalline cellulose, but not on amorphous substrates. This
study suggested that the leading tryptophan residue might act as
a recognition site for acquiring cellulose chains in a crystalline
context. von Ossowski et al. first made brief reference to similar
results being discovered for TrCel7A, but no data were
reported.461 In their initial report on HS-AFM, Igarashi et al.
demonstrated that the TrCel7A W40A mutant, wherein the
leading tryptophan residue in the −7 subsite is converted to
alanine, did not slide on crystalline cellulose.558 This result was
later corroborated in a more in-depth HS-AFM study from
Nakamura et al.575 In 2013, two computational studies, one
included with HS-AFM results from Nakamura and co-
workers575 and one from GhattyVenkataKrishna et al.,576 were
reported that shed further light on the role of Trp40 in chain
acquisition. Namely, both studies essentially simultaneously
reported that a cellononaose chain was placed in silico with the
leading glucose residue bound to Trp40; the chain advances by a
cellobiose unit into the CD to bind to the −5 to −7
subsites.575,576 In the aforementioned computational and
experimental study of TrCel7A and TrCel6A on cellulose,
MD simulation of the full length enzymes on the surface of
cellulose demonstrated that, in both enzymes, the entrance
tryptophan residue interacted directly with the first glucose
being decrystallized from the substrate.393
It is also noted that, in some GH7 CBHs, there is an aromatic
residue in the A1 loop that structurally resides on the opposite
side of the conserved tryptophan residue in the −7 subsite. For
example, in a ligand-bound structure of LqCel7B, the tyrosine
residue at this position was shown to bind to the planar face of
the glucosyl moiety bound in the −7 position.383 A computa-
tional examination of the HirCel7A CBH structure, which also
exhibits a tyrosine residue on the A1 loop, suggested similar
binding behavior.449
Beyond the entrance tryptophan residue, no experimental
mutation work has been reported, to our knowledge, for GH7
cellulases on the equivalent Trp38, Trp376, and Trp367 from
TrCel7A. Computational investigation of both Cel7A and
Cel7B has provided some clues as to how these residues might
relate to enzymatic processivity,577 as likely does analogy to
work in other GH families. In a computational investigation,
Taylor et al. found that mutating these aromatic residues to
alanine resulted in reduced binding affinities in all cases, but they
did so to different extents between TrCel7A, a CBH, and Cel7B,
an EG. The mutational effects were fairly localized in Cel7A,
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similar to results found in TrCel6A,578 but they seemed to be
completely deleterious to ligand binding in the EG Cel7B.577 In
related GHs, Horn et al. and Zakariassen et al. published two
seminal studies on GH18 chitinases wherein aromatic mutations
closer to the catalytic center of the enzymes resulted in dramatic
impacts to processivity, but yielded significantly higher activities
on chitosan, a soluble form of chitin.563,579 The authors of those
studies suggested that aromatic residues in GH tunnels likely are
vital for processive action, but that processivity comes at a cost
in enzyme performance. For GH7 cellulases, clearly, further
experimental work will be required to fully elucidate the role of
the aromatic residues in enzyme tunnels and clefts.
Advanced molecular simulation methods have also con-
tributed to the formation of a molecular basis for CBH
processivity. Payne et al.580 utilized free energy perturbation
with replica-exchange MD to develop a connection between
structure and processivity. The binding free energy, ΔGob, of
several family 7 CBHs was calculated, and a novel theoretical
connection was made to experimentally observable quantities:
ΔG bo ⎛ P intrk ⎞
= ln⎜ on
⎟
RT ⎝ kcat ⎠
In a comparison of the binding free energy of TrCel7A with a
tunnel-spanning cellononaose ligand to that of a celloheptaose
filling only the reactant side of the tunnel, it was calculated that
filling the product sites results in stronger binding by 11.1 kcal/
mol.580 Normalized by the number of binding sites, the product
sites bind the ligand more tightly than the substrate sites. This
exceptionally strong binding in the product sites of CBHs is
likely a key factor in driving processive motion. Following
product expulsion, the product sites are empty, and the cellulose
chain must advance forward by one cellobiose unit (Figure 35).
This study revealed the significant stabilization experienced by
the enzyme-cellulose complex by filling the product sites.
Furthermore, tight binding of cellobiose in the product binding
sites is remniscent of the well-known phenomena of cellobiose
inhibition in TrCel7A.581,582 The value reported by Payne et al.,
−11.1 kcal/mol,580 is in excellent agreement with a prior study
by Bu et al., −11.2 kcal/mol,571 obtained using an entirely
different computational approach. This prior study highlighted
the likely relationship of favorable cellobiose product binding to
product inhibition. The effects of cellobiose inhibition on
processive cellulolytic action are described further in section 6.3.
Pingali et al.583 employed small angle neutron scattering
(SANS) in order to probe the effect of pH on the structure of
TrCel7A in solution. Their findings indicated that at higher pH
(7.0, 6.0, and 5.3) the enzyme shape is well-defined and
compact, consistent with the crystal structure.172 However, at
pH 4.2 (near the pH for optimal catalytic activity), the CD
adopts a conformation that is intermediate between this
compact form and a fully denatured state, while maintaining
its secondary structure. It was speculated that this may indicate
enhanced conformational flexibility between the secondary
structure elements that would allow increased access for binding
to the cellulose chain, thus explaining the optimum in catalytic
activity.
Molecular simulation has provided insight into the molecular
level roots of the pH-dependent, higher-level conformational
changes observed via SANS by Pingali et al. The typical protocol
for MD simulation involves fixing the protonation state of the
protein’s titratable residues based on the solution pH and the
residue’s pKa and local environment. However, changes in local
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environment and protein conformational changes could result in
protonation/deprotonation processes that will be missed in this
scheme. Bu et al.479 utilized constant pH MD (CPHMD) in
order to directly couple the protonation state of the titratable
residues of TrCel7A and TrCel6A to the solution pH
conditions. At pH of 5.0, near the pH of optimal activity, the
boat (TrCel7A) or skew boat (TrCel6A) conformations for the
−1 sugar are favored over the stable chair. Increased loop
flexibility, particularly in loops B2 and B3 (and to a lesser extent
A2 and A4), was observed in TrCel7A at pH 5 when compared
to pH 7 due to altered hydrogen bonding interactions. Similar
increased loop flexibility was seen in TrCel6A. These findings
have important implications for catalysis and chain processivity,
respectively. In addition, comparing the apo to the substrate-
bound enzyme revealed significant differences in the pKa values
of the active-site titratable residues.
Maupin and co-workers applied these techniques to the CBH
MaCel7B and determined that the active site was highly charge-
coupled.478 In particular, Asp214 and His228 are critical for
shuffling protons around the active site and maintaining the
catalytically active states of Glu212 (deprotonated) and Glu217
(protonated). Incorporation of the CPHMD and replica
exchange MD results into a kinetic model demonstrated that
charge coupling between Asp214, Glu217, and His228 was
essential to reproducing the experimental kinetic−pH profile.
Follow-up work using these tools examined the flexibility of
tunnel-enclosing loops as a function of solution pH.478 The
findings of Bu et al. were affirmed in that increased loop
flexibility with varying pH is the likely source for the pH-
dependent morphology seen in the neutron scattering studies.
6.3. Product Inhibition
While strong binding at the product sites of CBHs may be a key
factor in driving processive motion, it also has an undesired
consequence for cellulose hydrolysis: product inhibition. There
is great potential for inhibition of enzyme cocktails due to the
exceptionally heterogeneous nature of lignocellulosic hydrolysis,
which involves a complex substrate as well as multiple
components of the enzyme cocktail. However, the most
dramatic inhibition of cellulases is due to the reaction products,
namely cellobiose and glucose. Product inhibition retards the
overall conversion rate of lignocellulose to the end product
glucose and is particularly nefarious at the high substrate
loadings utilized industrially.584,585 Additionally, product
inhibition has been considered as a factor in the rapid rate
retardation that occurs at short time and low conversion.586−588
Various reviews have included discussion of product inhib-
ition,32,33,556 with a recent excellent two-part review focused
exclusively on the topic.584,589 Both TrCel7A (CBH) and
TrCel7B (EG) are inhibited by cellobiose, and this likely
originates from their relatively high binding affinities for
cellobiose. As GH7 CBHs constitute the majority of industrial
cellulase mixtures, product inhibition constitutes an important
consideration for achieving high product yields in the enzymatic
hydrolysis of cellulose,556,584 which can have significant impact
on the efficiency of biomass conversion.590,591 Though other
strategies exist for relieving product inhibition in cellulases,
including product removal via membrane filtration585,592 and
conversion via cellobiose dehydrogenase (CDH),593 the most
commonly employed strategy is conversion of cellobiose to
glucose via β-glucosidases.594−597
Early work by Halliwell and Griffin598 on the activity of T.
koningii component C1 (CBH) showed that cellobiose inhibited
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Review
the hydrolytic activity on BC. At 79 μM cellobiose, only 40% of
the activity was retained; at 158 μM, all activity was lost. The
substrate concentration did not significantly affect measured
inhibition, and addition of β-glucosidase greatly increased the
solubilization of cotton fibers. Bacterial cellulases from C.
thermocellum were later shown to be inhibited by cellobiose and,
to a lesser extent, glucose.599 Addition of β-glucosidase from
Aspergillus niger was later shown to increase the glucose yield by
mitigating cellobiose inhibition.594 The authors suggest that a
combination of product and substrate inhibition causes the
detected retardation of hydrolysis at high substrate loading
(greater than 10%).
Lee and Fan studied the kinetics of the T. reesei cellulase
system on insoluble cellulose and the effect of extended
hydrolysis times.588 They suggested that the reduction in
hydrolysis rate may be caused by the inhibitory effect of the
formed products but also the transformation of cellulose into a
less digestible form of increased crystallinity. They also showed
that the cellulase cocktail is more strongly inhibited by
cellobiose than glucose. The product inhibition mechanism
was suggested to be deactivation of the substrate-adsorbed
enzyme and thus uncompetitive inhibition. However, Holtzap-
ple et al. estimated the binding of cellobiose to a cocktail of
cellulases from T. reesei and concluded that cellobiose inhibition
was noncompetitive.581 The authors discuss the possibility of a
regulatory site other than the active site. However, they
conclude that the binding of sugar inhibitors is likely at the
active site given that the active site is structured to strongly bind
polymeric forms of these sugars, the low diffusivity of the
insoluble substrate, and the stronger binding of cellobiose versus
glucose (because it has more sites of attachment). They also
compared the glucose and ethanol inhibition of the cellulases,
concluding that glucose inhibition was 1.4× greater than ethanol
inhibition, indicating that conversion of glucose to ethanol
effectively reduces the inhibition.
Väljamäe et al. detected cellobiose production by monitoring
the solution absorbance of a coupled reaction with CDH.600
They concluded that the early rate retardation is not because of
product inhibition by measuring initial cellulose hydrolysis rates
by TrCel7A both in the presence and in the absence of initial
cellobiose and finding that they follow an identical time
course.600 Furthermore, by adding fresh substrate to an already
slowed-down experiment produced a new “burst” phase
indicating that the retardation could not be due to
deactivation/inactivation of the enzymes themselves. These
conclusions were later affirmed for the bacterial EGs E2 (GH6)
and E5 (GH5), where addition of β-glucosidase did not
stimulate cellulose hydrolysis when measured on filter paper or
PASC.601
Though some early studies measured product inhibition with
crystalline substrates,602−605 many focused on small, soluble
substrates.461,606−610 Vonhoff et al. measured cellobiose
inhibition of TrCel7A on 2-chloro-4-nitrophenol-β-D-lactoside
to be Ki = 20 μM,608 a figure which has often been used for
comparison in subsequent literature. These findings contributed
to strong product inhibition being considered as a source of the
cellulose hydrolysis rate retardation seen at low conver-
sion.581,582 Gruno et al. studied product inhibition on one
CBH (TrCel7A) and three EGs (TrCel7B, TrCel5A, and
TrCel12A) from the T. reesei cellulolytic system via 3H reducing
end labeled BC and amorphous cellulose.611 They tracked short
time (5−10 s) radioactive product release in the presence of
varying concentrations of background cellobiose. With such a
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short time interval, one avoids the effects of continuous
alterations in cellulose structure due to hydrolysis as well as
the complex kinetics that may be relevant at later times, and thus
approximates a steady-state situation. Although the precise
nature of the inhibition (competitive or mixed) was not
determined, the relative strength of inhibition was calculated
(assuming a competitive paradigm, though assuming mixed
inhibition gives Ki values within the standard error of one
another). The value for the apparent competitive inhibition
constant found for TrCel7A on 3H-labeled BC was approx-
imately 1.5 mM,611 or 2 orders of magnitude higher than what
had been found for the same enzyme on small, soluble
substrates.461,607,608 Thus, product inhibition is actually
decreased 100-fold on the more industrially relevant solid,
insoluble substrates. Product inhibition for two of the EGs
studied (TrCel7B and TrCel5A) was about an order of
magnitude weaker than for TrCel7A611 (consistent with what
had been found previously on small molecule substrates607,612
and later confirmed on PASC613), and may be due to the more
open binding site cleft of EGs. An anomalous trend was found
for EG TrCel12A wherein activity actually increased with
cellobiose concentration suggesting this EG is activated by the
product; however, this is more likely an experimental artifact
due to enhancement of transglycosylation.611 A simple kinetic
model was also presented wherein the type of inhibition
experienced by the enzyme was found to be dependent upon the
relative binding strengths of the nonproductive (i.e., product
sites empty) and productive (i.e., binding tunnel-spanning)
enzyme−substrate complexes. Competitive inhibition results
when the productive complex is bound much more tightly than
the nonproductive; conventional mixed-type inhibition results
when the opposite is true. This was a landmark study toward
determining the relevance of product inhibition for the
industrial production of fuels and chemicals via enzymatic
hydrolysis of lignocellulosic biomass.
Bommarius et al. presented a study of the hydrolysis of MCC
(Avicel) prepared via three different methods of pretreat-
ment.596 Product inhibition mainly affected the hydrolysis rate
during “phase I” of hydrolysis, up to about 30% substrate
conversion and when the hydrolysis rate is highest. In the later
phases when the hydrolysis rate has slowed down, other factors
such as “jamming” of enzymes molecules on the substrate were
suggested to be rate-determining.
In a study of the synergism between endo- and exo-acting
cellulases on 14C-labeled BC, Jalak et al. studied cellobiose
inhibition557 under both single-turnover and steady-state
conditions and concluded that the inhibition of hydrolysis rate
by cellobiose was stronger under steady-state than under single-
turnover conditions. The product was mainly competing with
the cellulase chains to bind to TrCel7A by reducing the number
of initiations. Cellobiose also seemed to slow down the
processive movement of the CBH on the cellulose chain. This
was expected since cellobiose bound in the product site should
restrict the possibility of a processive movement and should
contribute to a noncompetitive component of the inhibition.
However, the calculated values of kinetic constants suggest that
the expulsion of cellobiose should not be rate-limiting for the
turnover. Additionally, the kinetic analysis led to the suggestion
that cellobiose also can bind in the substrate sites in direct
competition with the cellulosic substrate. Thus, both com-
petitive and noncompetitive components of inhibition should be
involved in cellobiose inhibition of TrCel7A resulting in an
overall mixed type of inhibition. The same group later utilized
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similar methods in thermophilic/thermotolerant fungi (A.
thermophilum, T. aurantiacus, and C. thermophilum) to
demonstrate that GH7 CBHs were more sensitive to product
inhibition than were GH6 CBHs and EGs.614 Also, cellobiose
inhibition decreased as temperature increased. The inhibition
measured using 14C-labeled BC did not correlate with that
measured using low molecular-weight model substrates
suggesting that the latter should not be used to measure
product inhibition as a parameter for selecting enzymes for
lignocellulose conversion557 and affirming what was concluded
by Gruno et al.611 and later by Murphy et al.615 The latter study
compared inhibition by cellobiose and glucose for the five major
cellulases from T. reesei and confirmed previous findings that
TrCel7A was most sensitive to cellobiose.
Structural studies of cellulases (section 6.2) have provided
some links to the molecular-level origins of the phenomenon of
product inhibition. Becker et al. attempted to engineer TrCel7A
with regards to its pH optimum by analogy with HiCel7B, which
has a higher and broader pH optimum than either TrCel7A or
TrCel7B.328 HiCel7B has an extra histidine residue near the
catalytic acid/base that the T. reesei enzymes do not have. Five
point mutations were made to TrCel7A, adding this histidine as
well as changing nearby residues to accommodate the bulkier
histidine residue (formerly an alanine). The primary goal of
shifting to a more alkaline pH optimum was achieved as well as
an unexpected additional consequence: cellobiose inhibition was
greatly relieved in the mutant CBH (Ki increased from 20 μM to
755 μM, both on 2-chloro-4-nitrophenol-β-D-lactoside sub-
strate). In addition, inhibition in the wild-type was competitive,
whereas, in the mutant, it was mixed. This is all the more
interesting when one considers that the “binding pose” of the
cellobiose in the product sites as well as the directly contacting
ligands of the enzyme are nearly identical in the wild-type and
the mutant. However, the weaker binding was attributed to the
loss of two water-mediated interactions between cellobiose and
Thr226 and Asp262. von Ossowski et al.461 presented another
protein engineering study wherein the exo loop of TrCel7A was
mutated in various ways to connect its structure with catalytic
function. One mutant in particular had eight residues deleted
from the exo loop, which significantly relieved inhibition by
cellobiose (as well as changing its type from competitive to
mixed). Ki was measured to be 24 μM for the wild-type TrCel7A
on pNPL and 300 μM on the exo loop deletion mutant with
substrate 2-chloro-4-nitrophenol-β-D-lactoside. This study also
considered PcCel7D (Ki = 180 μM on 2-chloro-4-nitrophenol-
β-D-lactoside), which has an intermediate length exo loop owing
to the natural deletion of six residues. The authors pinpoint
structural roots for the relative inhibitory strengths of these
three enzymes. The deletion mutant loses carbohydrate
interactions with Tyr247, Thr246, Tyr252, and Arg251 (residue
numbering for TrCel7A). However, PcCel7D maintains the
Arg251 residue (Arg240 in PcCel7D), which has hydrogen
bonding interactions with both of the product site glucosyl
residues. Further comparative studies of TrCel7A and PcCel7D
examined the binding of several inhibitors in addition to
cellobiose.458 It was observed that the loss of hydrogen bonding
interactions along with the more open binding tunnel represents
the structural basis for weaker binding of cellobiose to PcCel7D,
and thus reduced product inhibition.458,461 The more open
tunnel provides more access to solvent and more potential
disruption of protein−carbohydrate electrostatic interactions.
Textor et al. presented the crystal structure and inhibition
experiments on ThCel7A noting that a key difference between
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this enzyme and TrCel7A is in the exo loop mobility, as
discussed in section 6.2.466 The exo loop itself is quite similar to
that of TrCel7A, but its mobility is enhanced due to a missing
interaction with Tyr371 (TrCel7A numbering) from the loop
on the opposite side of the binding tunnel (loop A3). This
increased loop mobility was intimated by increased temperature
factors in the crystal structure for this region and confirmed with
MD simulations. This difference may be the key to explaining
why this enzyme’s inhibition by cellobiose is significantly
reduced, with a Ki = 7.2 mM on pNPC that is more than 2
orders of magnitude greater than that of TrCel7A on 2-chloro-4-
nitrophenol-β-D-lactoside.
Molecular simulation has served to connect structural roots of
product inhibition with thermodynamics. Bu et al. calculated the
absolute binding free energy of both glucose and cellobiose in
the product sites of TrCel7A. Using two different computational
methods, they found the absolute binding free energy of
cellobiose to be −14.4 kcal/mol (via steered MD) and −11.2
kcal/mol (via free energy perturbation MD) signifying that
cellobiose is 11.2−14.4 kcal/mol more stable in the TrCel7A
product sites than in solution.571 In order to pinpoint specific
residues that contribute to this strong binding, the product site
residues that interacted most strongly with cellobiose in the
simulations (Arg251, Asp259, Asp262, Trp376, and Tyr381)
were mutated to alanine, each resulting in significantly
weakened cellobiose binding. The same group followed up on
this work to elucidate how product binding differs between
processive and nonprocessive cellulases as well as how the
presence of a ligand in the substrate binding sites (−7 to −1)
affects the binding strength.572 Absolute binding free energy
calculations with GH7 (CBH TrCel7A and EG TrCel7B) and
GH6 (CBH TrCel6A and EG H. insolens Cel6B) revealed that
cellobiose binding in the product sites is dramatically stronger in
CBHs than in EGs lending a thermodynamic basis to what had
been observed experimentally.607,611−613 This result was
rationalized in terms of the structural differences between EGs
and CBHs: the more open architecture of the binding cleft in
EGs leads to increased solvation, thus weakening the protein−
carbohydrate interactions in the product sites. Payne et al.580
found via an independent computational method that filling the
product sites of TrCel7A results in an 11.1 kcal/mol
stabilization580 (in agreement with the results from Bu et
al.571). This serves as another confirmation of the exceptionally
strong binding that is present in the product sites in CBHs,
which is likely a key factor in product inhibition. Theoretical and
kinetic modeling studies have also helped to shed light on the
reaction kinetics, mechanisms, and enzymatic synergism of
product inhibition on enzymatic hydrolysis (refs 581, 582, 588,
591, 600, 602, 605, 609, and 616−619), and this body of
literature has recently been reviewed.584,618
Beyond cellobiose product inhibition, cellulase cocktails face
other complicating inhibitory factors. For example, many other
species besides the cellobiose product exist in the hydrolytic
system which also inhibit the GH enzymes, including lignin,620
ethanol,616,617 glucose,581,614 lactose,458,606 various ions,458,598
solvents (including ethanol, butanol, and acetone),581 hemi-
cellulose-derived sugars (including mannose, galactose, and
xylose),587,621 xylan and xylooligomers,622 and long (DP 7−16)
xylo- and gluco-oligosaccharides produced during pretreatment
(which were shown to inhibit T. reesei CBHs 100x more strongly
than cellobiose).623 Cellobiose is a stronger inhibitor than each
of these with the exception of the last.623 In addition, the
common industrial technique for product inhibition relief
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Review
(addition of β-glucosidases) is complicated by the fact that β-
glucosidases themselves are inhibited by glucose585,591,624 and
by gluconic acid.625 β-Glucosidase inhibition by gluconic acid is
particularly significant because the LPMOs that have become an
indispensable part of industrial cellulase cocktails produce
gluconic acid in significant amounts.626,627 In addition, β-
glucosidases have transglycosylation capabilities628−631 wherein
they assemble di- and oligosaccharides rather than deconstruct
them, a phenomenon that is exacerbated as glucose concen-
trations increase.
6.4. Pyroglutamate
Another common feature of GH7 enzymes, and other secreted
proteins from, e.g., T. reesei, is an N-terminal modification of a
glutamic acid residue to pyroglutamate, also referred to as
pyrrolidone carboxylic acid or PCA (Scheme 2 and Figure 40)
Scheme 2. Pyroglutamate Chemistry: The Chemistry of N-
Terminal Glutamine Cyclization
(refs 172, 309, 323, 329, 436, 438, 567, and 632−634). This
modification was known before the first GH7 crystal structures
were solved due to the nonstandard signatures on mass
spectrometry observed during N-terminal sequencing.436,438,632
The enzyme responsible for this chemical modification is
glutaminyl cyclase.635,636 This post-translational modification is
thought to be responsible for protection against degradation by
exo-acting peptidase enzymes.637,638 Similarly, the removal of
this post-translational modification is well-known via a calf liver
pyroglutamate amino peptidase, a method that has been
commonly used for preparing eukaryotic proteins for N-
terminal sequencing since the 1980s.639 GH7 structures to
date have been derived from fungal expression hosts, and as
mentioned above, pyroglutamate is ubiquitously observed in
these structures. However, the role of this post-translational
modification was only recently reported in the academic
literature.640 Motivated to understand the significant differences
in GH7 activity and stability imparted by heterologous
expression in S. cerevisiae, Dana et al. expressed T. emersonii
(R. emersonii) Cel7A from S. cerevisiae and Neurospora crassa
expression with a linker and CBM taken from A. thermophilum
and Agaricus bisporus, respectively.640,641 Improper disulfide
bond formation in S. cerevisiae and the extent of hyper-
glycosylation were both examined, and reported not to be a
major factor in the activity differences. Specifically, free thiols
were not detected, suggesting that all cysteine residues were
paired, and the yeast expression system used exhibits a deletion
in the glycosylation pathway resulting in minimization of
hyperglycosylation. Further deglycosylation by EndoH and a
mannosidase enzyme resulted in an increase in activity by 25%,
but did not fully explain the disparity in activity between the
fungal and yeast expressed GH7 enzyme, which led the authors
to examine pyroglutamate (Scheme 2). Treatment of the S.
cerevisiae-expressed Cel7A with glutamyl cyclase in vitro resulted
in essentially identical activity and stability to the enzyme
expressed in N. crassa.640
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Figure 40. N-Terminal pyroglutamate exemplified by TeCel7A (PDB code 3PFJ). The ligand from the TrCel7A Michaelis complex is shown in
aquamarine “sticks” (PDB code 4C4C).

6.5. Glycosylation
GH7 glycosylation significantly affects activity and represents a
promising target for protein engineering efforts. Glycosylation is
a post-translational modification that serves a myriad of
biological functions in recognition and signaling.642,643 Secreted
carbohydrate-active enzymes are often “decorated” by N-linked
and O-linked glycosylation, wherein small molecule carbohy-
drates are covalently attached to specific sites on the protein. N-
glycosylation attaches the carbohydrate (generally highly
branched mannose or single N-acetylglucosamine residues
(GlcNAc)) to the β-amide group of an asparagine in a N−X−
S/T motif (where X ≠ P), and O-glycosylation attaches the
carbohydrate (generally one to three mannose residues) to the
β-hydroxyl group of a serine or threonine.369 Glycosylation of
CBMs and linkers is discussed in sections 5.1 and 5.2,
respectively. In what follows, we focus our attention the
glycosylation of the catalytic domain (CD) of GH7 cellulases.
Early work by Maras et al. characterized six dominant forms of
N-glycans on TrCel7A produced from the RUT-C30 strain.644
Six major forms were characterized: GlcMan8 GlcNAc2,
GlcMan7GlcNAc2, Man7GlcNAc2, ManPGlcMan7GlcNAc2, Figure 41. TrCel7A CD glycosylation. The CD of TrCel7A is shown in
GlcMan5GlcNAc2, and Man5GlcNAc2. Klarskov et al. success- green transparent “surface”, N-glycans are show in slate and red
fully identified three sites for glycosylation in TrCel7A (out of “sticks”, and the cellulose surface is shown in aquamarine and red
four putative N-glycan attachment sites).456 Liquid chromatog- “sticks”. N-glycosylation attaches to the TrCel7A CD at 3 locations:
raphy coupled electrospray ionization mass spectrometry (LC- Asn45, Asn270, and Asn384. Blue squares denote GlcNAc, and green
ESMS) results indicated that a single GlcNAc residue was circles denote mannose. Both images show a representative glycan at
attached at Asn45, Asn270, and Asn384. The relatively small each binding site, though variability has been observed experimentally,
glycan prompted speculation that the glycan had been trimmed as discussed in the text.
by an endoglycosidase. The three sites identified in this early
study have been subsequently confirmed as the primary actually possessed attached sugar residues.368 They also
attachment sites for N-glycosylation on the TrCel7A CD demonstrated via monosaccharide analysis that only single
(Figure 41).369 For example, Harrison et al. analyzed a particular GlcNAc residues attached at these sites. O-glycosylation of GH7
strain of T. reesei, ALKO2877, and confirmed via ESMS that CDs has not been reported to date, in contrast to the linker and
only three of the four postulated TrCel7A N-glycosylation sites CBM (see section 5).368,645
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Hui et al. examined TrCel7A from RUT-C30 and two
derivative strains via MS and found that single GlcNAc residues
were predominantly found at Asn45 and Asn384 for all three
strains, but high mannose chains tended to attach at Asn270
with a strain-dependent degree of variability.273 The Asn270-
linked glycan was identified as Man8GlcNAc2 in RUT-C30,
single GlcNAc in one derivative strain, and a mixture of the two
in the other derivative strain. These differences may be due to
differences in the presence of glycan-trimming glycosidases,
which vary with strain and growth conditions. This may also be
the source of the shorter (and more consistent) length of
glycans attached to Asn45 and Asn384: perhaps steric hindrance
prevents glycosidases from accessing Asn270 glycans to the
extent that they do at Asn45 and Asn384. Follow-up work by
this group on TrCel7B revealed Asn56 (single GlcNAc residue)
and Asn182 (Man8GlcNAc2) as glycan-attachment sites for this
EG.418
The dependence of the glycosylation pattern of TrCel7A on
growth conditions was probed systematically by Stals et al.419
Four different growth conditions were examined: minimal
medium (resulting in a solution pH of 2.5), corn steep liquor-
enriched medium (pH 5), CaCO3-supplemented minimal
medium (pH 7), and fed-batch cultivation (pH 4). Differences
in growth conditions were shown to affect the number of
glycosylation sites that were occupied (ranging from zero to
three), attached glycan (Man5GlcNAc2 to Man8GlcNAc2),
glucosylation (i.e., a glucosyl moiety attached to the terminus
of the glycan, e.g., GlcMan8GlcNAc2), phosphorylation (e.g.,
ManPGlcMan8GlcNAc2), and frequency of single GlcNAc
attachment. Minimal medium tended to give a higher degree
of glycan occupancy than rich medium. Phosphorylation and
terminal glucosylation did not trend discernibly with the growth
medium conditions. The observation from Hui et al.273 that
Asn270-linked glycans tend to be more resistant to hydrolytic
trimming was affirmed.
Stals et al. also studied the effect on glycosylation produced by
different strains, all under minimal medium growth con-
ditions.646 Wild-type strain QM6A, RUT-C30 mutant, and
four other high-cellulase producing mutants (RL-P37, QM9414,
VTT-D-80133, and VTT-D-78085) were compared. RUT-C30
and RL-P37 (both selected using ultraviolet light and 2-
deoxyglucose-supplemented media) produced longer glycans
(GlcMan7−8GlcNAc2) whereas the wild-type and other mutants
produced trimmed glycans (Man5−6GlcNAc2), which may
indicate an inefficient glucosidase in the endoplasmic reticulum
in the former two strains. Interestingly, these two strains secrete
3−5 times more total cellulase than wild-type QM6A or
QM9414,647 though the connection between extended glycans
and cellulase production was not directly elucidated.
Jeoh et al. expressed TrCel7A in A. niger, resulting in 6-fold
increased N-glycan attachment to the CD.645 The increased
glycosylation resulted in a concomitant decrease in activity (on
both bacterial MCC and PASC) and increase in nonproductive
binding (as compared to homologously expressed TrCel7A).
The original activity could be restored by supplementing with
N-glycosidase that trimmed back the glycans. The decrease in
activity and binding relative to homologous TrCel7A were less
dramatic (though not eradicated) when the CBM and linker
were proteolytically cleaved. The recombinant CD displayed a
reduced binding affinity compared to homologous TrCel7A,
indicating that the glycans may sterically hinder its attachment
to the substrate surface (and thus account for the reduced
activity).
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A complementary study by Adney et al. utilized site-directed
mutagenesis to individually manipulate the glycan attachment
sites on the Cel7A CD from T. reesei and Penicillium
f uniculosum.648 Subsequent expression in A. niger and character-
ization revealed all mutants with glycosylation removed (two for
TrCel7A and three for Pf Cel7A) exhibited improved cellulose
conversion. The N384A mutant for TrCel7A, however, had a
much more dramatic effect than did the N270A mutant,
increasing the activity by 70%. This residue resides on a
substrate-binding tunnel loop and is near to the cellulose surface
when the enzyme is bound. This was significant in that it linked
glycosylation function with the enzyme structure and attached
glycans. In addition to three mutants that removed glyco-
sylation, an additional mutant for Pf Cel7A (A196S, creating an
N−X−S motif) introduced a new glycosylation attachment site
at Asn194. This increase in glycosylation actually gave the most
dramatic improvement (85% increase in activity) for any of the
Pf Cel7A mutants. Thermal stability also tended to be reduced
for the mutants versus the wild-type, both for addition and
removal of glycosylation.
Gao et al. utilized homologous expression of four glycoforms
of P. decumbens Cel7A to probe N-glycosylation at two
attachment sites, Asn137 and Asn470.508 They found
ManxGlcNAcy residues (where x = 2−5 and y = 1−2) attached
at Asn137. The glycoform with the lowest amount of mannose
in its glycosylation was shown to have zero detectable activity on
pNPC, but it was only this glycoform that synergistically
increased the glucose yield of an industrial enzyme cocktail (by a
factor of 2). Site-directed mutagenesis of Asn137 and Asn470 to
aspartate (thus removing the attachment glycan sites) resulted
in higher activities (the double mutant had 65% higher activity
than the most active of the four glycoforms).
These studies have demonstrated that it is possible to
characterize and manipulate glycosylation to alter cellulase
activity. Looking to the future, studies that can elucidate the
underlying structural features and interactions that give rise to
the observed differences in enzymatic function (e.g., hydrolytic
activity and thermal stability) will be particularly valuable for
harnessing glycosylation for improved cellulases.
6.6. Protein Engineering
As mentioned multiple times, GH7 enzymes are often the most
prevalent members of fungal cellulolytic cocktails and provide a
significant amount of hydrolytic potential. Thus, unsurprisingly,
they have received significant attention in academic, govern-
ment, and industrial research laboratories for engineering higher
activity, higher thermal stability, and advantageous modification
of other properties. However, a major problem in the
engineering of GH7 cellulases, especially GH7 CBHs, is the
use of a reliable expression host that is able to properly fold and
glycosylate the enzymes such that the activity and stability are
comparable to those expressed in filamentous fungi. To date, it
seems that yeast such as S. cerevisiase is able to express certain
GH7 CBHs such as TeCel7A (albeit still at low levels compared
to filamentous fungi) but not others such as TrCel7A.289 The
sequence relationships that give rise to this nonuniform
expression level are unknown but are essential to understand
in order to develop high-throughput expression systems for
GH7 CBH expression and screening. Nevertheless, significant
efforts in the open literature either using rational design or
semirandom mutations identified through computational
methods have demonstrated that GH7 engineering is feasible
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to date, as briefly reviewed here. We note that we do not review
the patent literature in detail here.
Early work in GH7 CBH engineering from Becker et al.328
and Boer and Koivula327 demonstrated that it is possible to
rationally shift the pH optimum of TrCel7A toward a more
alkaline optimum through site-directed mutagenesis. This pH
optimum shift was based on comparison of the TrCel7A (CBH)
active site to the HiCel7B (EG) active site, the latter of which
contains a histidine residue near the acid/base residue (Glu217
in TrCel7A), which corresponds to an alanine residue in
TrCel7A. To accommodate the bulky histidine residue in place
of alanine in the TrCel7A active site, four additional mutations
were conducted, resulting in an enzyme with a basic pKa shift of
approximately +0.7 pKa units, but a lower kcat/KM overall
compared to the wild-type on 3,4-dinitrophenyl-β-D-lactoside.
Boer and Koivula subsequently examined the wild-type and the
same pentamutant TrCel7A to understand the enzyme stability
as a function of pH. They demonstrated that the pentamutant
was less stable overall than the wild-type at both acidic and
alkaline pH, suggesting that mutations to shift pH optima
should go hand in hand with mutations aimed at improving
thermal stability.327
Voutilainen and co-workers from VTT have also published an
extensive series of studies to improve the thermal stability of
GH7 CBHs via several different methods.381,504,505,649 In a study
from 2007, they used a high-throughput robotics system to
generate a library of random mutants of MaCel7B expressed in
S. cerevisiae, and screened activity as a function of temperature
on 4-methylumbelliferyl-β-D-lactoside.504 The authors found
that a single mutation in the hydrophobic core of the enzyme
(S290T) was able to improve the Tm of the enzyme by 1.5 °C
and was able to double the activity of the enzyme on Avicel at 70
°C compared to the wild-type. In 2009, Voutilainen et al.
described a subsequent engineering study using MaCel7B
wherein the S290T mutation was combined with the addition of
a tenth disulfide bridge near the tunnel entrance (positionally
analogous to the tenth disulfide bridge in the TrCel7A CD), to
produce a 4.5 °C increase in Tm.505 The authors also employed
the strategy of expressing MaCel7B with the TrCel7A CBM-
linker domain attached, which resulted in a 2.5 °C increase in
Tm. Comparison of the MaCel7B activity to TrCel7A (both
enzymes were studied as both full-length enzymes with CBM-
linker and as solitary CDs) demonstrated that, at 45 °C,
TrCel7A in both cases was more active, but MaCel7B was
significantly more active at 70 °C, also in both cases. The lower
overall activity of MaCel7B at lower temperature was attributed
to a higher degree of product inhibition. Nevertheless, these two
studies demonstrated the ability to engineer a GH7 CBH for
higher stability and activity through a combination of both
random and rational mutagenesis.504,505
The same group from VTT also examined means to improve
the thermal stability of another thermophilic GH7 CBH, namely
TeCel7A, expressed in S. cerevisiae.649 Therein, they used a
computational prediction tool to predict 5 new sites for
engineering in disulfide bonds, all near the active site tunnel.
Like MaCel7B, TeCel7A natively contains 9 disulfide bonds, and
the addition of 3 independent disulfides increased the Tm in the
range 3.5−5 °C each. The combination of all three successful
engineered disulfide bonds increased the Tm by 9 °C. Two single
disulfide bond mutants and a triple mutant with all three
disulfide bonds added improved the activity on Avicel at 75 °C,
and the optimal triple mutant was able to hydrolyze Avicel at 80
°C with only slightly reduced performance relative to its activity
1362
Review
at 75 °C, thus demonstrating that the addition of disulfide bonds
can improve the thermal stability of GH7 CBHs.
Arnold and co-workers from the California Institute of
Technology have also made substantial contributions to GH7
CBH engineering primarilyon the basis of computational
prediction tools such as structure-guided recombination.650−652
In 2010, Heinzelman et al. used five GH7 CBH parent enzymes
to predict and express (in S. cerevisiae) 28 chimeric GH7 CBHs,
on the basis of a “background” structure of the TeCel7A enzyme,
chosen due to its high expression levels in yeast.650 The authors
found multiple combinations of mutations (with an average of
37 mutations relative to the closest parent enzyme) that resulted
in substantial thermal stability improvements. Moreover, activity
on MCC for 6 chimeras was slightly retained at higher
temperatures than the parent enzymes (68, 70 °C). Interest-
ingly, activity at 37 °C in 6 chimeras was improved, also on
MCC, but the overall conversion time was quite short (90 min)
when the relative activity measurements were made. The same
group reported a follow-up study in 2012 from Komor et al.651
starting with 5 chimeras from the previous work. Therein, they
used a method based on GH7 CBH sequence alignments and
the FoldX force field to predict the effect on ΔGfolding for each
amino acid residue. Using this approach, the authors were able
to demonstrate an improved variant with 8 additional stabilizing
mutations that exhibits a T50 of 72.1 °C, which is a substantial
increase in thermal stability. This mutant also retained
significant extent of activity on MCC up to 70 °C. Via yet
another method, namely noncontiguous recombination from
the same group, Smith et al. located 6 single amino acid
mutations that are each able to improve the TrCel7A stability by
1−3 °C each.652 Overall, these studies from Arnold et al.
demonstrate that computational protein design tools combined
with large screening studies can identify both single-point
mutations and blocks of enzymes that can directly contribute to
improved stability.
High-throughput, random mutagenesis has also proven to be
effective, given a reliable secretion system. Dana et al. reported
the development of an S. cerevisiae strain capable of producing
GH7 CBHs with limited glycosylation at consistent titers.641
Using this system, they developed a library of GH7 CBHs with
biased clique shuffling starting with 11 parent Cel7A genes, 86%
of which were active. Overall, 51 chimeras were identified with
improved thermal stability, and several were shown to have
activity on Avicel hydrolysis significantly higher than TeCel7A at
60 and 65 °C.
6.7. Conclusions
Our collective understanding of GH7 cellulases to date is quite
considerable, especially given recent developments toward more
quantitative underpinnings of their action on cellulose. In
summary, the following important features of GH7 cellulases
have been elucidated:
(1) GH7 cellulases employ a two-step, retaining mechanism,
and CBHs and EGs form two distinct populations that
differ primarily in their loop structures near the ligand.
(2) GH7 CBHs can engage cellulose chains via exo-initiation
or endo-initiation, i.e., binding to chains by the end or via
internal binding to chains.
(3) The rate-limiting step in GH7 CBH action in the absence
of synergistic enzymes is likely to be substrate dissociation,
either caused by obstacles or amorphous regions of
cellulose.
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Figure 42. TrCel6A structure and active site details. (A) The overall structure of the TrCel6A CD from PDB code 1QK2, showing the distorted β/α
barrel, with labeled structural elements.194 (B) The TrCel6A structure rotated to show the active site tunnel enclosed by an N-terminal and C-terminal
loop (pink and blue cartoon, respectively). (C) Four of the TrCel6A binding sites (−2 to +2) revealed by the cocrystallized nonhydrolyzable o-
iodobenzyl-1-thio-β-D-cellobioside ligand (cyan stick). Aromatic residues are shown in magenta stick, and residues that have been hypothesized to
participate in catalysis are shown in green stick. The water molecules likely involved in catalysis are shown as red spheres.

(4) The rate-limiting step in GH7 CBH action in the
presence in the presence of synergistic enzymes is likely is
the processive velocity (i.e., the combined steps of
hydrolysis, product expulsion, and processive motion).
Synergistic enzymes, especially those that provide
endolytic action, likely provide points of detachment for
GH7 CBHs, thus removing the rate limitation of substrate
dissociation.
(5) Computational studies of the entire GH7 CBH processive
cycle suggest that the glycosylation step in hydrolysis is
the rate-limiting step between hydrolysis, product
expulsion, and processive motion.
(6) The CBM-linker domains on GH7 cellulases seemingly
do not significantly contribute to the enzyme perform-
ance once the enzyme is productively bound to the
substrate, but likely play a major role in enzyme targeting,
and thus impact the overall enzyme performance.
Although GH7s represent one of the most well studied (if not
the most well studied) classes of cellulolytic enzymes to date,
there are many open questions based on our current
understanding of their action. Given their significant presence
in industrial cocktails combined with their powerful hydrolytic
action, engineering these enzymes is of keen importance for
industrial biomass conversion. The development of accurate
structure−activity relationships for the GH7 cellulases still
remains nascent.
One of the primary challenges in studying these enzymes is
expression in convenient heterologous expression systems,
which severely limits high-throughput production of adequate
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amounts of enzyme for biochemical, structural, and enzyme
performance studies. Slowly emerging tools in specialized yeast
strains, engineering of filamentous fungi, and cell-free expression
systems for these enzymes, combined with a deeper under-
standing of the effects of post-translational modifications on
activity and stability, will eventually enable an era wherein rapid
screening of GH7 cellulases from natural diversity and
engineered enzymes will be possible. The recent publication
of the effect of pyroglutamate clearly established its importance,
if not yet the mechanism by which it functions, in enzyme
activity and stability.640 Clearly, a more comprehensive
understanding of GH7 glycosylation is now of paramount
importance, an area which has been only slightly reported on to
date in the academic literature given the complexity of detailed
glycan analysis.369
On the basis of quite recent results, it seems that the
glycosylation step in the retaining mechanism is the primary rate
limitation of GH7 CBHs. If this conclusion is correct, which
seems logical given the strength of glycosidic bonds, then the
primary target for GH7 improvement is kcat. Certainly, future
kinetics studies similar to that of Kurašin et al. in the presence of
additional cellulolytic enzymes (e.g., GH6 cellulases, additional
EGs, β-glucosidases, and LPMOs) on a broader range of
substrates will continue to inform the direct target for GH7
improvement. Furthermore, the development of additional,
more utilitarian methods for studying processivity of GH7
cellulases in the presence of additional enzymes will be of
paramount importance for the systematic comparison of GH7
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Table 8. Reported GH6 Crystal Structures

source and original name in PDB resolution
primary citation code (Å) brief highlights ref
Fungal CBH Structures
Trichoderma reesei CBHII/ 3CBH 2.00 first GH6 structure reported; cocrystallized with a nonhydrolizable ligand (o-iodobenzyl-1-thio-β- 192
Cel6A D-cellobioside)
1CB2 2.00 Y169F mutant 574
1QK2 2.00 wild-type with (Glc)2-S-(Glc)2 194
1QJW 1.90 Y169F mutant with (Glc)2-S-(Glc)2 194
1QK0 2.10 wild-type with m-iodobenzyl-β-D-glucopyranosyl-β(1,4)-D-xylopyranoside 194
1HGW 2.10 D175A mutant 656
1HGY 2.20 D221A mutant with β-D-glucose 656
4AU0 1.70 D221A mutant with 6-chloro-4-methylumbelliferyl-β-cellobioside 704
4AX6 2.30 D221A mutant with 6-chloro-4-phenylumbelliferyl-β-cellobioside 704
4AX7 1.70 D221A mutant with 4-methylumbelliferyl-β-cellobioside 704
Humicola insolens Cel6A 1BVW 1.92 659
2BVW 1.70 binding of glucose in the −2 subsite and cellotetraose in +1 through +4 subsite confirms the 662
existence of 6 subsites
1GZ1 1.90 D416A mutant with (Glc)2-S-(Glc)2 209
1OCB 1.75 wild-type complexed with fluoresceinylthioureido-derivatized tetrasaccharide 665
1OC6 1.50 D405N mutant 665
1OC5 1.70 D405N complexed with 4II-thio-β-cellotetraoside substrate 665
1OC7 1.11 D405N complexed with methyl-4,4II,4III,4IV-tetrathio-α-cellopentoside substrate 665
1OCJ 1.30 D416A complexed with methyl-4,4II,4III,4IV-tetrathio-α-cellopentoside substrate 665
1OCN 1.31 D416A complexed with cellobio-derived isofagomine 666
Coprinopsis cinerea Cel6C 3A64 1.60 670
3ABX 1.40 wild-type complexed with p-nitrophenyl-β-D-cellotrioside 670
3A9B 1.20 wild-type complexed with cellobiose 670
3VOF 1.60 D102A complexed with glucose 671
Coprinopsis cinerea Cel6A 3VOG 1.45 wild-type complexed with HEPES 671
3VOH 2.40 wild-type complexed with cellobiose 671
3VOI 2.00 wild-type complexed with p-nitrophenyl-β-D-cellotrioside and with a Mg2+ ion in the active site 671
3VOJ 2.29 D164A 671
Chaetomium thermophilum 4A05 1.90 wild-type complexed with cellobiose and cellotetraose with a Li+ ion in the active site 672
Cel6A
HJPlus chimera Cel6A 4I5R 1.50 chimera of H. insolens, T. reesei, and C. thermophilum 674
3C6P chimera Cel6A 4I5U 1.22 chimera of H. insolens, T. reesei, and C. thermophilum 674
Fungal EG Structure
Humicola insolens Cel6B 1DYS 1.60 663
Bacterial CBH Structures
Thermobif ida f usca E3/Cel6B 4B4H 1.50 wild-type 683
4B4F 2.20 wild-type complexed with cellohexaose (chain A) or cellotetraose (chain B) 683
4AVO 1.80 D274A complexed with cellohexaose 686
4AVN 2.00 D226A/S232A complexed with glucose and cellotetraose 686
Bacterial EG Structures
Thermobif ida f usca E2 (Cel6A) 1TML 1.80 this apo structure was virtually identical to the previously reported fungal GH6 CBH, but lacked 658
one of the tunnel-enclosing loops
2BOD 1.50 wild-type complexed with (Glc)2-S-(Glc)2 688
2BOE 1.15 Y73S mutant 688
2BOF 1.64 Y73S complexed with cellotetraose 688
2BOG 1.04 Y73S complexed with (Glc)2-S-(Glc)2 688
Mycobacterium tuberculosis 1UP0 1.75 wild-type complexed with cellobiose 667
H37Rv Cel6
1UP3 1.6 wild-type complexed with (Glc)2-S-(Glc)2 667
1UOZ 1.10 wild-type complexed with thio-cellopentaoside 667
1UP2 1.9 wild-type complexed with cellobio-derived isofagamine 667

enzyme performance across substrates and in cocktails of
varying composition.
7. FAMILY 6 GLYCOSIDE HYDROLASES
GH6 was one of the original six families identified through
hydrophobic cluster analysis.439 Originally denoted family B
GHs, T. reesei CBH II and two EGs from C. f imi and
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Streptomyces were identified as sharing over 20% sequence
identity. The tertiary structure of these enzymes was unknown
at the time, but only a year later, TrCel6A would be the first
reported cellulase structure providing critical insight into both
the general structure of this family and key modes of CBH
action.
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Figure 43. Sequence alignment of the six fungal GH6 structures reviewed here. Strictly conserved residues are shown in red block, and chemically
similar residues in red text. The blue boxes indicate chemical similarity across a grouping of residues. The secondary structural elements of TrCel6A are
shown above the sequences. The catalytic acid is marked by a yellow star. Residues with hypothesized or confirmed roles in catalysis are marked by
magenta stars. Active site loops A and B are marked by black boxes. The figure was generated with ESPript (http://espript.ibcp.fr).347

By 1991, membership of this family had grown by one to
include Microbispora bispora EG A.654 Family B was renamed
family 6, reflecting the growing availability of sequence data.654
Recent sequence analysis suggests GH6 enzymes may be further
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categorized into eight subfamilies with accessory loop lengths
defining the features of the different groups.655 Today, GH6
encompasses nearly 500 protein sequences from both bacteria
and eukaryota remaining one of the smaller designated
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families;151 we stress that this by no means diminishes the value
of this family in industrial biomass conversion.
Members of the GH6 family have been experimentally
characterized as exhibiting two primary modes of action, endo-
hydrolytic cellulose action (EC 3.2.1.4) and exo-hydrolytic
cellulose action (EC 3.2.1.91), where many are thought to
display both to some extent. Ståhlberg et al. proposed that all
GH6s can create new chain ends in cellulose as an EG would,
and thus, none are purely exoglucanases.304 While cellulolytic
action in general is critical to industrial biomass conversion,
GH6s play a particularly unique role by virtue of their
complementary mode of action. The GH6 family is currently
the only known family having cellulases that act from the
nonreducing cellulose chain end.151 The most effective enzyme
cocktails contain cellulases that recognize both the reducing and
nonreducing ends allowing rapid, synergistic degradation of
crystalline content.305,436,476,540 As such, GH6 cellulases are
primary components in biomass degradation cocktails.
In addition to their industrial relevance, GH6s have been
critical instruments in the general study of cellulase action.
TrCel6A was first uncovered in the characterization of the
culture filtrate components from the enhanced QM 9414
Trichoderma strain,435 though the enzyme would not explicitly
be named until 1980 as CBHII.436 Several years later, Teeri et al.
isolated and sequenced the CBHII gene adding a second
sequence by which to compare CBHI (TrCel7A).325 The
solution of the T. reesei CBHII CD 3-D structure marked a
major turning point in cellulase research.192 Biochemical
characterization prior to this had successfully identified CBHII
as a modular enzyme consisting of a CD and a CBM.344,399
Additionally, key details regarding the CBHII mode of action
had been uncovered including that the enzyme acts processively
from the nonreducing end of cellulose and that it yields
primarily cellobiose through an anomeric inversion stereo-
chemical pathway.302,443 With the solution of the first GH6
crystal structure, details regarding processive action began to
emerge.192 In addition to revealing the overall enzyme typology
and active site location (Figure 42), the CBHII structure solved
by Rouvinen et al. uncovered a central question motivating
many subsequent structural and biochemical studies, namely the
lack of a readily identifiable catalytic base, which is reviewed at
length here. As we note below, although there was significant
debate for many years, there is now a general consensus that the
GH6 catalytic mechanism proceeds via a “water wire” or
Grotthuss mechanism potentially with or without an explicit
catalytic base on the enzyme, although this still lacks definitive
confirmation.573,656 Over time, CBHII would receive its current
designation of Cel6A (TrCel6A), which we use throughout the
remainder of our discussion.657
In this section, we describe our current understanding of
fungal GH6 cellulase structure, function, and efforts to engineer
thermal stability and activity in these important enzymes. We
describe the hypothesized catalytic mechanism and the
structural and biochemical studies that support the proposed
mechanism, the many studies describing processive action and,
to a lesser extent, the synergism studies. As bacterial GH6
cellulases often provide interesting and insightful comparison to
fungal GH6s, we occasionally deviate from the primary focus of
the review on what we hope is an informative detour. A
summary of discussed GH6 structures is provided in Table 8.
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Review
7.1. Structural Studies
7.1.1. TrCel6A: Wild-Type. The first cellulase structure
revealed the catalytic core of TrCel6A and the general GH6 fold
as a distorted β/α barrel (PDB code 3CBH192). Much like the
classic (β/α)8 barrel, this structure consists of a core region of β-
strands surrounded by a constellation of α-helices. The noted
distortion arises from the exclusion of one of the eight β-strands
from within the core barrel region. The C-terminal loop
connecting the β7 strand to the α8 helix (shown in blue in Figure
42B) and the loop connecting the β2 strand to the α4 helix
(shown in pink in Figure 42B) form the enzyme active site. For
convenience, we have labeled these loops as loop A and loop B,
respectively, shown on the multiple sequence alignment in
Figure 43.
The active site region of TrCel6A is a 20 Å-long tunnel
formed by two loops, loops A and B.192 The loops partially
encompass the cellulose substrate and several water molecules.
In subsequent structures, the flexibility of these loops would be
elucidated as well as the potential relevance in nucleophilic
attack (Figure 42B).194 The enclosure somewhat restricts the
tunnel volume and thus the substrate flexibility. This latter
observation is consistent with previously observed CBH product
profiles, as substrate rearrangement to a productive binding
conformation is prevented when the chain is advanced by a
single glucose moiety.302 Rouvinen et al. proposed that the
tunnel shape is typical for processive attack of cellulose chains
and suggested the loops surrounding the active site appear to be
ideally positioned to assist in this processive action.
In addition to uncovering the fold of GH6, the TrCel6A
structure illustrated how the enzyme binds a cello-oligomer
within the tunnel-shaped active site. Cocrystallization with o-
iodobenzyl-1-thio-β-D-cellobioside inhibitor identified four
substrate binding subsites, which Rouvinen et al. labeled A−
D; the structure has not been deposited in the PDB. Current
nomenclature refers to these sites as −2 through +2 (Figure
42C).175 The binding subsites are largely defined by the spatial
arrangement of aromatic residues within the active site. The
thio-oligosaccharide complex illustrated carbohydrate-π stacking
interactions with three tryptophan residues (Figure 42C).
The TrCel6A structure also enabled an initial proposal for the
GH6 catalytic mechanism. At the time of the Rouvinen et al.
study, GH6 enzymes were known to invert substrate stereo-
chemistry,443 yet candidate residues corresponding to this
mechanism had not been definitively identified. The new
structure showed two carboxylate oxygens belonging to Asp175
and Asp221 located less than 5 Å from the glycosidic bond
between the −1 and +1 subsites.192 Rouvinen et al. proposed
that this location is the cleavage site and that these two residues
likely participate in catalysis. Specifically, the authors put forth
the hypothesis that Asp221 was protonated, and thus the likely
catalytic acid, and that Asp175 was charged aiding in
protonation of Asp221 as necessary. A water molecule in
position to attack the anomeric carbon at the cleavage site was
also captured (Figure 42C). Identification of a residue acting as
the catalytic base proved less straightforward, with the authors
offering two potential candidate residues, Asp263 or Asp401.
This latter observation spawned many subsequent studies and
controversy, and definitive identification of the catalytic base, if
one indeed is required, still remains elusive.
As we continue to discuss GH6 structure and catalytic
function, it is helpful to compare subsequent structures
providing insight by analogy. Figure 43 provides a sequence
alignment of TrCel6A alongside several of the more relevant
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GH6 sequences. The active site loops as well as residues of
particular interest to understanding the catalytic mechanism
have been highlighted.
7.1.2. T. f usca Cel6A. The first EG representative from
family 6 was solved just a few years after TrCel6A and proved
useful in identifying topological differences between EGs and
CBHs. Spezio et al. solved the structure of T. fusca Cel6A
(designated E2 at the time; PDB code 1TML).658 Although a
bacterial EG sharing only 26% sequence identity with TrCel6A,
the topologies of the two enzymes are quite similar. As is
characteristic of the GH6 fold, Tf Cel6A also displays the
distorted β/α barrel, though with different α-helix lengths. A
notable difference between the two structures is the topology of
the active site. Tf Cel6A is missing a homologous N-terminal
loop, which yields a more open cleft-shaped active site
potentially to allow flexibility in endo-initiated attack of
crystalline cellulose (Figure 44). On the surface, the active site
Figure 44. Comparison of the fungal CBH TrCel6A (1QK2)194 and
bacterial EG Tf Cel6A structures (1TML).658 The TrCel6A structure is
shown on the left in gray surface. The thio-oligosaccharide ligand is
shown in cyan stick. Active site loops A and B are shown in pink and
blue, respectively. The Tf Cel6A structure is shown on the right in
green surface. For illustration, Tf Cel6A is shown bound to the thio-
oligosaccharide from the TrCel6A 1QK2 structure. The Tf Cel6A active
site is notably more open than the enclosed tunnel-shaped active site of
TrCel6A as a result of shorter and missing loop regions. The cleft- or
groove-shaped active site is a common attribute of many EGs.
structural differences between Tf Cel6A and TrCel6A are
consistent with the notion of a strict delineation of endo-
versus exo-initiated attack; though as noted before, it is known
that CBHs are also capable of endo-initiation despite having a
tunnel-shaped active site,304 making such a delineation based on
topology alone not definitive.
The Tf Cel6A structure confirmed several suppositions from
Rouvinen et al. regarding catalytic residues while casting doubt
on others.192 Spezio et al. noted that a homologous pair of
aspartates was in a similar location to Asp175 and Asp221 in
TrCel6A.658 Asp117 (Asp221 in TrCel6A) was likely proto-
nated given its relative distance to neighboring residues and was
reported as the catalytic acid on this structural basis. However,
Spezio et al. suggested that the Asp79 (Asp175 in TrCel6A) was
unlikely to participate in catalysis, as it shifted away from the
active site by 4 Å. Notably, the Tf Cel6A structure did not
contain a ligand, which may have allowed a greater degree of
active site flexibility. Spezio et al. did make the concession that
the loop containing Asp79 may shift upon binding.
Similarly, Spezio et al. examined the identity of the catalytic
base on the basis of structural data. Focusing on the aspartate
homologous to TrCel6A Asp263 (Asp156/Tf Cel6A), they
found it was unlikely this residue participated in catalysis as the
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Review
catalytic base. The Asp156 carbonyl oxygen was buried in the
active site, though still within hydrogen bonding distance of the
catalytic acid. Spezio et al. suggested Asp156 more likely
functions to modulate pKa of the catalytic acid. The other
proposed catalytic base, Asp401 in TrCel6A (Asp265), was
observed forming a salt bridge with a neighboring arginine.
Spezio et al. suggested Asp265 was readily ionizable and
adequately positioned to accommodate both the ligand and
attacking water molecule in the inverting stereochemical
mechanism; thus, the hypothesis from this study was that
Asp265 serves as the catalytic base in GH6s.658
7.1.3. TrCel6A: Y169F Variant. With an unclear picture of
the residues involved in catalytic function, Koivula et al. set out
to examine the role of a centrally located tyrosine residue
(Tyr169) in catalysis and substrate binding.574 The authors
suggest Tyr169 sterically hinders the glucan substrate in the −1
binding site, resulting in formation of the catalytically active
conformation. The Y169F variant was generated and solved in
its apo form (PDB code 1CB2). This new structure displayed
negligible structural differences relative to the original wild-type
structure. Weighing in on the ongoing debate as to catalytic
residues, Koivula et al. contradicted proposals that Asp401 or
the Tf Cel6A homologue could act as the catalytic base. The
authors argued that Asp401’s participation in two salt bridges
would likely prevent it from extracting a proton from the
attacking water. Turning attention to Asp175, Koivula et al.
confirmed the previous structural interpretation indicating that
the residue is important for pKa modulation and ensures
protonation of the catalytic acid.192,574 This observation was
arrived at on the basis of unpublished mutagenesis studies.
Additionally, the authors noted that Asp175’s position could
potentially serve to stabilize an oxocarbenium-like transition
state.
Ultimately through structural evidence and specificity and
kinetic characterization, Koivula et al. arrived at the hypothesis
that Tyr169 serves to distort the −1 subsite glucose moiety
positioning the glycosidic linkage for catalysis. Comparisons of
catalytic constants toward short cello-oligosaccharides were
examined for both the wild-type and the Y169F variant, and kcat
for cellotriose and cellotetraose were 4 times lower for the
Y169F variant. Specificity constants were also decreased. This
indicates Tyr169 plays a key role in catalysis. The Y169F
mutation was also accompanied by an altered pH activity profile
leading one to speculate that Tyr169 may also play an indirect
role in ensuring protonation of the catalytic acid.
7.1.4. H. insolens Cel6A: Apo. The solution of the catalytic
core of H. insolens CBH Cel6A (HiCel6A) and subsequent
characterization cast doubt on its categorization as a strictly exo-
enzyme (PDB code 1BVW).659 HiCel6A is strikingly similar to
TrCel6A, sharing 64% sequence identity. Thus, one may
reasonably presume the two enzymes share a great deal of
characteristics related to specificity, kinetics, and processive
action. Up to this point, TrCel6A had been largely described as
an exo-active CBH. However, several groups were approaching
the conclusion that an enzyme, but this one in particular, may
not be strictly delineated into either the endo- or exo-
category.304,660 Varrot et al. observed hydrolysis of a
fluoresceinyl-derivatized oligosaccharide substrate by HiCel6A,
the functional group of which appeared far too large to reside in
the closed, tunnel-shaped active site of the HiCel6A structure.
The authors argued that, to accommodate the fluorescein group,
the active site of HiCel6A must necessarily exhibit flexibility in
the active site loop regions. Flexibility of these loops would in
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theory allow for endo-initiation on cellulose, explaining more
recent observations indicative of such.209
The catalytic acid, Asp226, was also observed in two
orientations, perhaps representing two different protonation
states (Figure 45).659 In one conformation, the distance
Figure 45. Two observed conformations of the HiCel6A catalytic acid
likely correspond to two different protonation states. The 1BVW
structure is shown in green cartoon. The catalytic acid, Asp226, and the
proposed catalytic base, Asp405, are shown in stick. The distance
between the two residues in one conformation is consistent with a
single-displacement catalytic mechanism, and thus, Asp405 (Asp401/
TrCel6A) was not excluded from consideration on the basis of distance.
between the carbonyl oxygen of the proposed catalytic acid
and the proposed catalytic base is 9.5 Å (Asp401/TrCel6A and
Asp405/HiCel6A), which is consistent with a typical single-
displacement mechanism. Varrot et al. further agreed with the
hypothesis that an aspartate serves to modulate the pKa of the
catalytic acid, as Asp268 was appropriately positioned to do so.
However, while the HiCel6A Asp180 is in an equivalent position
to Asp175 of TrCel6A, the authors reported their consideration
of its relevance to catalysis as inconsequential on the basis of its
distance from the cleavage site. They cite consistency with
mutagenesis studies performed on similar enzymes as further
support of this conclusion.192,661
7.1.5. H. insolens Cel6A: Cello-Oligomer Complexes.
Shortly after solution of the original HiCel6A apo structure,659
Varrot et al. reported a glucose/cellotetraose-bound structure of
HiCel6A confirming their previous hypothesis that the active
site loops are flexible, changing conformation upon substrate
binding, and providing significant molecular-level insight into
ligand binding (PDB code 2BVW).662 The noted conforma-
tional change in the active site loops leads to a more enclosed
active site tunnel that increased the number of contacts with the
ligand. This likely also applies to the TrCel6A active loops
indirectly explaining previous observations that TrCel6A is able
to access and hydrolyze internal bonds.304 Direct observation of
this phenomenon in TrCel6A would soon follow,194 and
computational studies would later define molecular mechanisms
associated with the conformational change.571
Six ligand-binding subsites, −2 through +4, were directly
observed as a result of these new oligosaccharide complexes.
The −1 subsite was unoccupied, and the glycosyl units in the
occupied subsites adopted a relaxed 4C1 conformation. Addi-
tionally, the catalytic acid, Asp226, was observed in close
proximity to the +1 glucose and within a realistic distance of
Asp405, again not ruling it out as the catalytic base. Varrot et al.
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Review
further suggest that in one models the missing −1 glucose
moiety, Asp405 would be a suitable distance from the −1
anomeric carbon so as to activate a water molecule for
nucleophilic attack. The authors maintained their opinion that
Asp180 significantly impacts the pKa of the catalytic acid given
the large conformational change about the Cα−Cβ bond
allowing Asp180 to hydrogen bond with the glucose.
7.1.6. TrCel6A: Non-Hydrolyzable Ligands. A set of
three new ligand-bound complexes of TrCel6A was instrumen-
tal in addressing the issue of loop flexibility upon substrate
binding.194 These structures included the following: wild-type
complexed with (Glc)2-S-(Glc)2 (PDB code 1QK2), Y169F
complexed with (Glc)2-S-(Glc)2 (PDB code 1QJW), and wild-
type complexed with m-iodobenzyl-β-D-glucopyranosyl-β(1,4)-
D-xylopyranoside (PDB code 1QK0). Zou et al. analyzed the
observed active site loop conformations of these three new
structures in the context of the available structures at the time.
The authors found that the conformational states of the active
site loops generally fall into four categories including “most
closed”, “more open”, “even more open”, and “most open”. The
tunnel-forming loop was responsive to modifications in the
active site including ligand binding as well as site-directed
mutagenesis (Y169F, D175A, and D221A). The former three
conformations are illustrated in Figure 46. Clearly, the TrCel6A
active site is quite flexible with its “pincer-like” narrowing of the
tunnel likely playing a significant role in the enzyme’s mode of
action.
Additionally, the crystallization of TrCel6A in the presence of
nonhydrolyzable ligands was effective in capturing TrCel6A with
the −1 glucose moiety in the 2SO distorted conformation
Figure 46. Flexibility of the TrCel6A active site loop A as observed in
four separate structures. The “most open” active site observed via
structural studies was captured in the 1HGW structure, shown in yellow
cartoon.656 The 1QJW active represents the “most closed” active site,
dark teal cartoon.194 Two intermediate active site loop conformations,
“more open” and “even more open”, were captured in 1QK2 and
1QK0, gray cartoon and magenta cartoon, respectively.194 The ligand,
cyan stick, is the thio-oligosaccharide linked ligand from the 1QK2
structure. For illustration, Ser181 is shown in stick on each of the loop
A conformations demonstrating the impressive range of motion of this
residue.
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providing additional insight in the ongoing efforts to define the
catalytic residues. In both the wild-type/(Glc)2-S-(Glc)2
complex and the Y169F complex, ligand binding in the active
site is essentially the same despite each having very different
active site loop conformations. The wild-type/m-iodobenzyl-β-
D-glucopyranosyl-β(1,4)-D-xylopyranoside complex captured
the −1 sugar in the 4C1 conformation; however, the xylosyl
unit of the ligand was in the −1 site with the glucosyl moiety in
the −2 site. Zou et al. note that if the −1 subunit sugar had a
hydroxymethyl group as in the native substrate, it would severely
clash with the Tyr169 side chain. The Y169F mutant also
captured the −1 glucose in a distorted conformation, suggesting
Tyr169 plays an indirect role in catalysis.194 Overall, these
findings suggest the −1 ring distortion arises from the need to
avoid steric clashes with the surrounding protein, yet the
distortion is likely integral to the catalytic mechanism.
Zou et al. also explicitly examined the three structures for
clues regarding the identity of the catalytic residues with respect
to existing characterizations and structural knowledge. Two
primary conclusions were made. The pair of aspartyl residues
previously proposed as catalytic acid and pKa modifying residue,
Asp221 and Asp175, respectively, serve as the primary catalytic
machinery, and neither of the putative catalytic bases, Asp263
and Asp401, are convincingly suitable candidates. With respect
to the first conclusion, Zou et al. suggested structural evidence
that pointed toward a catalytic mechanism that is dependent
upon the conformation of the active site loops: (1) In the most
commonly captured orientation corresponding to most apo
structures, the tunnel is generally closed, opening as necessary to
facilitate substrate entry. (2) When the substrate binds within
the tunnel, the −1 glucosyl moiety distorts, which is likely
crucial for catalysis. The aspartyl pair is locked in an interaction
with Tyr169 and Arg174 that facilitates the protonation of
Asp221. (3) A loop conformational change tightens the tunnel
breaking the Asp175/Asp221 interaction with Tyr169/Arg174
and positions two water molecules close to the −1 sugar of the
anomeric carbon. (4) The Asp175/Asp221 hydrogen bond is
broken to allow Asp221 to donate its proton to the substrate
leaving group, and Asp175 activates a neighboring water
molecule above the anomeric carbon. (5) Catalysis takes
place, and the α-anomer of cellobiose is expelled as product
alongside a final conformational change of the active site loops.
Processive action on cellulose would then follow.
Examining the putative catalytic bases, Zou et al. reported that
Asp263 is too distant to be directly involved in catalysis but may
modify pH behavior of the enzyme, as previously pro-
posed.658,659 Asp401 was not ruled out as the catalytic base by
distance and was likely to be in the correct charge state to
activate a water molecule. However, its side chain was not close
enough to a negatively charged residue and, thus, may not be
suitable to deprotonate the water molecule. Furthermore, the
Asp401 side chain does not interact with a suitable water
molecule in either of the thio-oligomer complexes but, instead,
hydrogen bonds with the −1 sugar O3. The placement of
Asp401 relative to the face of the sugar ring rules it out as the
catalytic base. As discussed below, subsequent reports suggest
that Zou et al.’s reported structures do actually indicate a
catalytic role for Asp401, though not as expected. Instead, it has
later been suggested that the backbone of Asp401 interacts with
the attacking water, and thus, this residue may be important for
proper alignment of the catalytic center.573,656
7.1.7. H. insolens Cel6B. The solution of the H. insolens EG
Cel6B (formerly EG VI) structure provided a unique
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perspective on the relationship of EG and CBH active site
topology with activity (PDB code 1DYS).663 Up to this point,
the tunnel-shaped active sites of CBHs defined by flexible active
site loops were largely thought to be a construct allowing
processive hydrolysis of crystalline cellulose.316,440 EGs were
identified through the lack of these active site loops resulting in a
groove- or cleft-shaped binding site. Deletion of one of the key
CBH active site loops in a C. f imi CBH indeed increased overall
activity toward CMC, but the activity toward smaller cello-
oligosaccharides was simultaneously decreased clouding the
notion that the absence of active site loops strictly delineates
activity toward soluble substrates.660
Adding to the suggestion that EGs may not always resemble
the canonical open active site architecture, Davies et al. reported
the structure of the HiCel6B, which displayed a striking
homology with family 6 CBHs.663 As with familial representa-
tives TrCel6A, Tf Cel6A, and HiCel6A, HiCel6B exhibits the
characteristic GH6 distorted β/α-barrel fold. However, HiCel6B
is missing one C-terminal loop (loop B) present in the CBHs
(Figure 43) creating a more open binding site, yet not
equivalent to other GH6 EGs. The authors noted that the
remaining N-terminal loop (loop A) may close upon substrate
binding allowing Ser98 (homologous to TrCel6A Ser181) to
interact with the −1 and/or +1 subsite substrate. Such a
conformational change could also allow Glu97 to interact with
the active site. Other EGs have a conserved histidine at the
Glu97 position, while CBHs exhibit an alanine.
In the ongoing search for the catalytic base, Davies et al. note
that Asp316 (homologous to TrCel6A Asp401) displayed salt
bridge to the conserved Arg269.663 Corresponding to findings
from Zou et al.,194 this configuration suggests Asp316 (and
TrCel6A’s Asp401) is an unlikely catalytic base in HiCel6B, but
the authors noted that a small rearrangement of the −1 subsite
sugar could better position the residue. Beyond the catalytic
base, Ala182 hydrogen bonds with the pKa modulator Asp180
that in turn orients the catalytic acid, Asp139, for catalysis.
7.1.8. H. insolens Cel6A: D416A/Thio-Oligosaccharide
Complex. With the identity of the catalytic base still unknown,
Varrot et al. set out to investigate a set of particularly perplexing
findings related to the proposed catalytic base in HiCel6A,
Asp405.209 The authors reported unpublished observations that
the EG HiCel6B is rendered inactive upon mutation of the
homologous residue (Asp316). However, mutation of Asp405
in the CBH HiCel6A resulted in only a 100−300-fold reduction
in activity rather than complete annihilation. These results led
the authors to propose that the CBHs of the GH6 family may
benefit from catalytic “rescue” as a result of the phenotypic
differences in architecture. One of the basic residues proposed as
a possible “rescue” residue in HiCel6A was Asp416. Varrot et al.
suggest that the residue sits in such a way that the Asp could
potentially activate a water molecule for inverting attack on the
−1 glucose anomeric carbon. Moreover, this residue is located
on a loop that is missing in homologous EGs and, thus, would be
a likely candidate to maintaining minimal levels of activity
should the potential base (Asp405) be modified.
Solution of a thio-oligosaccharide bound D416A variant of
HiCel6A was unable to confirm the hypothesis that Asp416
serves as a “rescue” residue because no water molecule was
observed in a position for nucleophilic attack (PDB code
1GZ1).209 Nonetheless, the D416A structure was successful in
capturing a thiol-linked cellotetraose molecule spanning the +2
to −2 binding subsites with the −1 glucose moiety in the 2SO
conformation. This structure was also very briefly one of the
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“most open” GH6 CBH active sites, in terms of position of the
two active site loops A and B. At this point in time, evidence
convincingly pointed toward distortion of the Michaelis
complex in the catalytic itinerary of retaining GHs; however,
it was unknown as to whether inverting GHs exhibited a similar
transition state distortion. Previous GH6 structures had
captured a similar 2SO conformation in the −1 binding
subsite,194 but the nearest potential transition state to the 2SO
conformation on the Stoddard diagram is the 2,5B boat
conformation, which initially seemed an unlikely transition
state. The D416A variant provided mounting structural
evidence of what appears to be a valid intermediate catalytic
state (2,5B) of inverting GH6s.
Overall, no significant structural changes were found for the
D416A mutant except for the open conformation of the active-
site loops. The authors report only a single direct hydrogen
bond between the −1 glucose O3 hydroxyl and the proposed
catalytic base Asp405; other protein−substrate interactions
appeared to be mediated entirely by water. During proof of the
manuscript, Varrot et al. included a postscript209 acknowledging
the findings of another paper published at nearly the same
time.656 Koivula et al. had solved the structure of an even more
open TrCel6A structure, but more importantly, they had
proposed a solvent-mediated Grotthuss mechanism for
deprotonation that was consistent with the solvent-mediated
interactions observed in the D416A HiCel6A structure.
Furthermore, Koivula et al. reported molecular simulations
that support the 2,5B transition state conformation.
7.1.9. TrCel6A: D175A and D221A Variants. The Koivula
et al. study in 2002 remains one of the most influential structural
investigations of catalytic residue function in GH6s.656 The
structure of TrCel6A D175A and D221A mutants (PDB codes
1HGW and IHGY, respectively) were solved, and activity
measurements, kinetic isotope experiments, and molecular
simulations were used to ascribe functions to Asp221 and
Asp175.656 A key finding of this study was that the catalytic
mechanism appears to proceed via water-mediated Grotthuss
mechanism (Figure 47), and that Asp175 may accept the proton
as the catalytic base.656 Moreover, the distortion of the substrate
in the −1 binding subsite was shown to be stable over a
relatively short MD simulation, suggesting the conformation is
stabilized by the surrounding protein environment. An addi-
Figure 47. Proposed GH6 Grotthuss mechanism. In this mechanism,
the role of catalytic base is fulfilled by a wire that shuttles the proton
from the attacking water.573 The residues are labeled according to the
TrCel6A sequence. The Ser181 and Asp401 backbone carbonyl
oxygens stabilize the attacking water, and the Asp175 side chain
stabilizes a second water molecule that will accept the proton from the
attacking water in an inverting mechanism.
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tional feature of note is that the D175A structure remains the
most open GH6 active site conformation yet observed.
The idea that Asp221 was the catalytic acid was not a new one
going into this study, nor was that of Asp175 as a pKa modulator.
Up to this point, most available data pointed toward Asp221 as
the catalytic acid.192,658,661 The D175A structure captured
Asp221 positioned close to where the −1/+1 glycosidic linkage
would be located (PDB code 1HGW). This position matched an
earlier structure of the Y169F TrCel6A variant which used a
thio-oligosaccharide to capture a substrate spanning the active
site (PDB code 1QJW).194 Using MD simulation to model the
oxygen-linked oligosaccharide, Koivula et al. observed that the
hydrogen bond between Asp221 and Asp175 breaks revealing a
new, catalytically competent side chain conformation of Asp221.
This conformation had been observed previously in Tf Cel6A
and HiCel6A structures but was the first observation of a
protonated carboxyl-oxygen oriented toward the glycosidic
linkage in TrCel6A. Activity measurements of the D221A
variant on cellotriose, cellotetraose, cellopentaose, and cellohex-
aose indicated a complete loss of competency; however, binding
specificity was maintained. The findings overwhelmingly
support Asp221 as the catalytic acid candidate.
Data supporting Asp175’s role in catalysis had been less
convincing up to this point. Koivula et al. began investigating the
function of Asp175 in catalysis by testing the natively expressed
D175A variant on oligosaccharides. They showed that the
variant was effectively inactivated but, like D221, maintained
binding specificity. The recombinantly expressed D175A variant
maintained 2−3% residual activity on barley β-glucan,
suggesting, as the authors state, that it is not the catalytic base
“in a normal sense”.656 The D175A variant also markedly
lowered the pH-rate profile, suggesting a role in pKa modulation.
The authors also put forth a convincing argument surrounding
the role of Asp175 in transition state stabilization. Examining
cellobiosyl fluoride substrate reaction kinetics, Koivula et al.
calculated an exceptionally high charge buildup in the TrCel6A
transition state. Electrostatic stabilization would be required to
maintain such an unfavorable ΔΔG⧧, and Asp175 is uniquely
positioned to perform this role. The structure captures the −1
glucose ring oxygen and the Asp175 carboxylate oxygen within
4.5 Å of each other with no other shielding atoms between them.
Many previous studies had proposed Asp401 as the mostly
likely candidate, but contradictory results prohibited conclusive
identification.661 Using structural insights and molecular
simulation, Koivula et al. laid out a rational discussion describing
the evidence against Asp401 acting as the catalytic base in
TrCel6A. They first described the role of Asp401 in catalysis and
why prior studies have been inconclusive at best. Structural data
indicate that Asp175 and Ser181 coordinate two water
molecules positioned so as to attack the −1 glucose anomeric
carbon. The water coordinated by Ser181 is also coordinated by
the backbone carbonyl of Asp401. Simulation of a carbocationic
intermediate confirmed this water molecule as the likely catalytic
water. The authors further added that prior studies mutating
Asp401 do not in fact remove the catalytic base but rather
induce a charge imbalance that destabilizes the transition state.
The removal of Asp401 would leave two nearby positively
charged residues without acidic compensation near the active
site.
Instead of a catalytic base-mediated mechanism, Koivula et al.
suggested proton transfer occurs through a water wire in a
Grotthuss-type mechanism, illustrated in Figure 47, wherein
proton transfer occurs to a neighboring water upon nucleophilic
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Figure 48. Proposed mechanism of catalysis and “virtual processivity” as captured through structural studies of HiCel6A bound to thio-oligosaccharide
inhibitors. The figure numbers referenced in the captions at right refer to the figures from the original publication. Reprinted with permission from ref
665. Copyright 2003 Elsevier.
attack, which may then transfer a proton to Asp175 serving as
indirect catalytic base.664 The authors noted that because there
is little evidence of bond formation with the nucleophilic water
in the transition state, there is likely no need for a traditional
catalytic base-mediated mechanism. Examination of solvent
isotope effects on kcat for cellotriose substrates supported the
active site architecture observed in the structural study, and thus
the proposed catalytic roles. Interestingly, a Grotthuss
mechanism for GH6 catalysis had previously been considered
in the 1999 review by Davies et al.170 Ultimately, the plausibility
of this mechanism had been dismissed as inconsistent with a
prior study claiming to have identified a GH6 catalytic base
residue.661
7.1.10. H. insolens Cel6A: D405N Variant. In 2003, Varrot
et al. revisited their structural investigation into the possibility of
a catalytic “rescue” residue upon mutation of the proposed
catalytic base, Asp405.665 The authors were searching for a
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structural explanation as to why mutagenesis of Asp405 results
in only a 100−300 fold decrease in activity rather than complete
abolishment, as in the H. insolens EG. A series of five structures
were reported toward the aim of this goal. These structures
included the following: wild-type HiCel6A complexed with
methyl 4-S-(4III-FTU-β-cellotriosyl)-4-thio-β-D-glucopyranoside
(PDB code 1OCB), an apo D405N mutant (PDB code 1OC6),
a D405N mutant complexed with methyl 4II-thio-β-cellotetrao-
side and methyl 4,4II,4III,4IV-tetrathio-α-cellopentaoside (PDB
codes 1OC5 and 1OC7, respectively), and a D416A mutant
complexed with methyl 4,4II,4III,4IV-tetrathio-α-cellopentaoside
(PDB code 1OCJ).665 The authors noted that there appear to be
relatively few structural consequences of mutating Asp405.
Neighboring residues rotate slightly but maintain a similar
interaction profile with the asparagine. Asp405 maintains a salt
bridge with the neighboring Arg357 as well, the destruction of
which was speculated to result in a significant local instability.
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Varrot et al. concluded that mutation of Asp405 must have a
participatory role in catalysis since the mutation does not result
in local structural unfolding. Prior studies, as well as this one,
noted that Asp405 is directly involved in substrate binding with
a hydrogen bond between the C3 hydroxyl group of the −1
glucose moiety. Additionally, it was proposed that Asp405 may
serve to stabilize the proposed 2,5B transition state intermediate,
though not directly observed as part of this study. The authors
again left open the possibility that Asp405 is the catalytic base.
The study of the D405N mutants bound to the unique set of
fluorogenic and thio-oligosaccharides also uncovered a series of
structures trapped in the act of “virtual processivity”.665 Varrot
et al. illustrated a proposed schematic of catalysis and
processivity in GH6 enzymes (Figure 48). The initial step,
productive binding mode, was captured in the wild-type
structure bound to the fluorescing thio-oligomer (1OCB).
Though the ligand does not span the +1/−1 binding sites where
cleavage of the glycosidic linkage would occur, the catalytic acid
reflects a catalytically competent conformation. This structure
also captured a ligand binding within the −3 and −4 subsites,
which had not been previously defined for any GH6 enzyme.
The authors suggested this finding illustrates that there is no
absolute requirement for cellulose chains to enter the enzyme
tunnel from one end only supporting the ability of this particular
enzyme to act in an endo-active fashion as well as exo-active. In
the productively bound conformation, the protein makes
approximately 15 different hydrogen bonds directly with the
ligand and a similar number of solvent-mediated hydrogen
bonds.
The start of the processive event, “processivity commences”
(Figure 48), was captured in the 1OC7 structure, the D405N
mutant bound to the thio-linked cellopentamer.665 The first
sugar lies intermediate to the −1/+1 binding site with the
trailing sugars also occupying intermediate subsite binding
positions as the chain threads in the tunnel. The interactions of
the protein with the ligand are similar to the productive binding
mode. The chain is hypothesized to progress through the tunnel
maintaining a nonproductive binding mode conformation. The
1OC5 structure of the D405N mutant bound to the thio-linked
cellotetramer illustrates the nonproductive binding mode in
which the ligand sits in the +1 through +4 binding subsites in a
relaxed chair conformation. Each of the glucose moieties’ faces is
rotated 180° from that of the productively bound ligand
conformation suggesting the ligand has partially completed the
processive motion through the tunnel. Contrasting the
productive binding mode, the protein makes very few hydrogen
bonds directly with the ligand in the nonproductive
conformation. Nearly all of the interactions are through
solvent-mediated hydrogen bonds as the ligand slides through
the tunnel. The catalytic acid is tilted away from the substrate.
This frequently captured dual orientation of the GH6 catalytic
acid, both of which were captured in this study, has long been
attributed to the change in local pH affecting protonation state.
Varrot et al. suggested that the conformational change may
actually serve a role in processivity, putatively to accommodate
the unusual orientation of the sugars as they find their way
toward a catalytically competent binding position. Overall, this
study documents a fascinating series of structures illustrating a
proposed processive mechanism and sheds light on the ability of
HiCel6A to act in endo-initiation mode.
7.1.11. H. insolens Cel6A: D416A/Isofagomine Com-
plex. Revisiting the H. insolens D416A variant, Varrot et al. used
a cellobiose-derived isofagomine inhibitor to successfully
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capture the 2,5B transition state conformation in the −1 binding
subsite (PDB code 1OCN).666 This was the first structural
report of this unusual catalytic itinerary, traversing from the 2SO
Michaelis complex conformation through the 2,5B transition
state intermediate.194,209 The structure reveals the distortion is
driven primarily through steric interactions with neighboring
Tyr174 coinciding with the hypothesized role of the
homologous residue in TrCel6A.574 The structure also
effectively supported the Grotthuss mechanism for GH6
cellulases. A water molecule was captured in position for attack
on the anomeric center of the −1 isofagomine moiety (Figure
49). The formerly proposed catalytic base, Asp405, was
Figure 49. Transition state intermediate of HiCel6A as captured in the
isofagomine bound complex with the D416A variant (PDB code
1OCN).666 Asp405, once proposed as the catalytic base, is shown
mediating the Grotthuss-type water wire mechanism through hydrogen
bonding of its main chain carbonyl with a water molecule. Ser186, of
loop A, also stabilizes the water wire. A second water molecule
hydrogen bonds with the first, and Asp180 completes the wire
interaction. Asp226, the catalytic acid, is positioned away from the pKa
modifying Asp180 during this intermediate state. Tyr174 forces the
energetically unfavorable 2,5B conformation of the −1 moiety through
steric interactions. This transition state intermediate appears to be
general for GH6 inverting enzymes.
captured mediating a water−ligand interaction by stabilizing
the interaction with the main-chain carbonyl. Ser186 on loop A,
the pKa modifying/indirect putative base Asp180, and a second
water molecule are also implicated in catalysis. This structure
unambiguously suggests GH6 catalysis occurs via a Grotthuss-
type water wire mechanism with Asp180 potentially being able
to accept a proton during catalysis (Figure 47).
Interestingly, the Grotthuss-type water wire mechanism may
be feasible even in the absence of a homologous serine residue in
loop A. Mycobacterium tuberculosis H37Rv Cel6, a bacterial EG,
exhibits an alanine substitution in this position. Structures of the
bacterial EG complexed with nonhydrolyzable substrates
indicate that, despite the alanine substitution, a water molecule
is still able to correctly position for inverting attack of the −1
moiety (PDB codes 1UP0, 1UP3, 1UOZ, and 1UP2).667 The
stabilization of the water molecule is alternatively maintained by
a conserved asparagine homologous to Asp401 in TrCel6A. This
latter study suggests that GH6 enzymes have evolved to
maintain the water wire catalytic mechanism.
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7.1.12. C. cinerea Cel6C. The genes encoding five new GH6
enzymes (CelA through CelE) from C. cinerea were discovered
in 2009.668 Of the five, only CcCel6A exhibits a CBM and was
most closely related to that of TrCel6A.669 The remaining GH6
cellulases were more similar in homology to HiCel6B. Liu et al.
confirmed that C. cinerea Cel6A (CcCel6A) exhibits a CBM,
while CcCel6B and CcCel6C do not. Yoshida et al. claim that all
five GH6s exhibit CBH activity on the basis of cellobiose
production from PASC. This latter observation became
increasingly more pertinent upon the solution of the structure
of CcCel6C in the year that followed.
Liu et al. reported the solution of the CcCel6C structure in
complex with cellobiose and p-nitrophenyl-β-D-cellotrioside,
which appears to be the first report of a GH6 from a
basidiomycete (PDB codes 3A64, 3ABX, and 3A9B).670 The
authors suggested the structure was an additional example of a
GH6 CBH based on prior characterization on PASC despite the
high sequence homology with the H. insolens EG Cel6B.
However, in the current study, CcCel6C was tested for activity
on CMC and demonstrated specificity for the substrate unlike
any other GH6 CBH. The new structure suggests the CcCel6C
active site is significantly more open than any of the known GH6
CBHs. Active site loops A and B, which are not missing (Figure
43), were captured in an open conformation in each instance
and appear to be insensitive to the substrate binding-based
conformational change noted in both TrCel6A and HiCel6A.
Accordingly, the serine residue of loop A (Ser181 of TrCel6A)
was not noted to interact with the substrate. A lysine
substitution in loop A is suspected for the reduction in flexibility
in the loops overall. Additionally, a tyrosine present in the −3
binding site of other GH6 CBHs is missing in CcCel6C making
for a much more open product-binding site by comparison.
Overall, the evidence that CcCel6C is indeed a CBH is not
compelling. In fact, specificity toward CMC and the appearance
of what was described as a cleft-shaped active site surrounded by
fixed open loops points more toward EG behavior, or at the very
least, a high degree of endo-initiation. High sequence homology
with the EG HiCel6B is also concerning. In lieu of assays on
more crystalline substrates such as Avicel or BMCC, the
categorization of CcCel6C activity as a CBH remains unclear.
7.1.13. C. cinerea Cel6A. In a subsequent study, Tamura et
al. investigate conformational changes within the active sites of
both CcCel6A and CcCel6C resulting from substrate binding or
mutation of the pKa modifying catalytic aspartate.671 Four
CcCel6A structures were reported including the following:
CcCel6A bound to 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES; PDB code 3VOG), CcCel6A bound to
cellobiose (PDB code 3VOH), CcCel6A bound to p-nitro-
phenyl-β-D-cellotrioside (PDB code 3VOI), and an apo
CcCel6A D164A variant (PDB code 3VOJ). A fifth structure
of an CcCel6C D102A variant with glucose in the −2 binding
site was also reported for comparison to the group’s prior wild-
type structure of CcCel6C.670
The solution of the CcCel6A structure was the second
basidiomycete GH6 structure reported. As noted before,
CcCel6A exhibits a CBM, whereas all other C. cinerea GH6s
do not. CcCel6A demonstrates 52% sequence identity with
HiCel6A and 48% identity with TrCel6A. Additionally, CcCel6A
was reported to produce cellobiose from PASC but not
hydrolyze either p-nitrophenyl-β-D-cellotrioside or CMC,
indicative of CBH activity. However, relative or specific activity
data has never been directly reported making direct comparisons
between CcCel6A and CcCel6C and to other GH6s difficult.
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Tamura et al. report the CcCel6A-HEPES structure maintains an
“open” tunnel conformation, while the CcCel6A-cellobiose and
CcCel6A-p-nitrophenyl-β-D-cellotrioside structures demonstrate
the “closed” form of the active site tunnel that now appears to be
characteristic of GH6 CBH loops upon substrate binding.194,671
The conformational changes in moving from open to closed
forms were essentially identical to changes observed in HiCel6A.
Interactions of CcCel6A with the bound ligands were reported
to be consistent with the Grotthuss-type catalytic mechanism
previously proposed for GH6s.656
Rather than the new CcCel6A structure, the primary focus of
the Tamura et al. study was actually the conformational changes
associated with mutation of Asp102 in CcCel6C.671 Reversing
their original conclusion that the active site of CcCel6C is rigid
by comparison to other GH6 CBHs,670 the authors noted that
the D102A mutation induces a significant degree of flexibility in
the active site loops. A “motion angle” measurement devised to
account for conformational changes indicates that CcCel6C
D102A variant active site loops tighten significantly in
comparison to both the wild-type active site and the CcCel6A
structures. Tamura et al. go on to suggest that the conforma-
tional change in the CcCel6C D102A variant is the most drastic
yet observed in a GH6 CBH. However, superimposition of the
two CcCel6C structures with the “most closed” and “most open”
extreme conformations from TrCel6A (1QJW and 1HGW,
respectively) indicates that the purported drastic conformational
change is only moderately greater than that of TrCel6A (Figure
50). The CcCel6C D102A variant exhibits loop structuring
nearly identical to that of the most closed TrCel6A structure,
1QJW. Overall, the more open active site of CcCel6C wild-type
again supports the enhanced ability of this particular GH6 to
perform endo-initiated attack relative to other GH6 CBHs.
Figure 50. Superimposition of CcCel6C structures with the TrCel6A
“most open” and “most closed” structures. The wild-type CcCel6C
structure, orange cartoon, represents the open conformation, which
Tamura et al. reported as the most drastic open conformation of a GH6
yet.671 The TrCel6A D221A variant in the “most open” conformation is
shown in yellow cartoon for direct comparison to CcCel6C.656 Upon
binding a glucose molecule, the CcCel6C D102A variant active site
loops close, shown in pink cartoon. This latter conformation is nearly
identical to the “most closed” conformation from the TrCel6A Y169F
variant, shown in teal cartoon.194 The thio-oligosaccharide ligand from
1QK2 is shown in cyan stick.194
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7.1.14. C. thermophilum Cel6A. Solution of the C.
thermophilum Cel6A structure provided additional structural
evidence in support of a general Grotthuss-type mechanism for
GH6 enzymes and again suggested GH6 enzymes have an active
site particularly suited for endo-initiated attack (PDB code
4A05).672 CtCel6A displays the typical GH6 distorted β/α
barrel including both full-length active site loops A and B.672
The enzyme shares 77% sequence identity with HiCel6A and
63% with TrCel6A suggesting it is a CBH. Specificity for BMCC
and filter paper supports this assessment.673 The wild-type
CtCel6A structure captured a cellobiose molecule in the −3 to
−2 binding subsites and a cellotetraose molecule in the +1 to +4
subsites. This is the second such account of a −3 binding site for
a GH6 enzyme and is suggestive of the ability of the active site to
bind substrate in a manner so as to allow glycosidic cleavage of
products other than just cellobiose.665 One of the more
interesting features of this structure was the tetrahedrally
coordinated Li+ ion located in the same position as the anomeric
carbon of the Michaelis complex. The Li+ ion likely mimics the
oxocarbenium-ion-like transition state complex coordinating
with three water molecules and the O4 hydroxyl of the +1 sugar,
consistent with a water-mediated inverting catalytic mechanism
in GH6 enzymes.
7.1.15. TrCel6A Variants HJPlus and 3C6P. Increasing
the ability of cellulases to withstand heat while maintaining a
reasonable degree of activity is an important area of industrial
enzymatic biomass conversion research. Wu et al. have been
particularly successful in identifying a set of mutations through
directed evolution that is capable of increasing T50 by nearly 20
°C over wild-type.674 To understand the structural implications
of these mutations and the molecular-level origins of added
thermal stability, Wu et al. solved the structures of the TrCel6A
thermally stable variants HJPlus and 3C6P (PDB codes 4I5R
and 4I5U, respectively).674 The thermal stability character-
ization efforts are discussed in greater detail in section 7.4, but
briefly, we mention that the HJPlus variant consisted of 48
mutations with respect to the wild-type TrCel6A enzyme. The
3C6P consisted of an additional 7 mutations with respect to
HJPlus, a total of 55 with respect to wild-type. Both variants
maintain a high degree of structural similarity with wild-type
TrCel6A. With so many mutations, the authors necessarily did
not discuss contributions from each mutation toward thermal
stability and focus primarily on residues providing stability gains
in 3C6P over HJPlus. Improvements in thermal stability were
attributed to five residues, all of which were located near the
surface of the enzyme. Mutations at the surface of globular
proteins are frequently capable of enhancing thermal
stability.675,676 These included the following: M135L, Q277L,
S317P, S406P, and S413P. Only the serine to proline mutations
were solvent exposed, however. The former two mutations
appear to improve thermal stability through improved hydro-
phobic interactions. In addition to proximity to the surface,
serine to proline mutations, as in the 3C6P variant, have long
been reported to contribute stability in loop regions by
restricting conformational freedom.677−679 In HJPlus, proline
substitutions appear to be well-tolerated, though they do not
always enhance stability. In fact, the three identified in the 3C6P
variant were the only serine to proline mutations to significantly
impact thermal stability in a positive fashion. While this
particular approach to engineering enhanced stability while
maintaining activity was effective, it is difficult to develop a
general principle by which other proteins may be modified to
the same effect.
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7.2. Catalytic Function
Structural and biochemical studies have been essential to
elucidate the catalytic mechanism of GH6 enzymes, although
many questions still remain. To date, these studies enabled a
general hypothesis for the unusual single-displacement catalytic
mechanism adopted by GH6s. In the section to follow, we
summarize our current understanding of the GH6 catalytic
mechanism including the roles of key residues in the active site,
the role of water in catalysis, aromatic residue function in
substrate binding, and conformational changes along the
processive cycle.
Four residues are well-conserved across GH6 enzymes: the
amino acids homologous to TrCel6A Asp221, Asp175, Asp263,
and Asp401 (Figure 43).655 Residues homologous to Asp221
putatively operate as the catalytic acid in conjunction with the
pKa modifying and potential “indirect” catalytic aspartate
Asp175. Several residues, including Asp263 and Asp401, have
been proposed as the catalytic base, though definitive evidence
remains elusive. Additionally, Tyr169 has been reported to play
a critical role in transition state stabilization. The function of
each of these residues is discussed below.
7.2.1. Catalytic Acid. Structural and biochemical character-
izations overwhelmingly suggest the GH6 catalytic acid is an
aspartic acid residue located to donate the proton to the
substrate leaving group as necessary. Rouvinen et al. observed
that TrCel6A is active over a pH range 4.0−7.0, a range that
allows both charged and uncharged aspartyl side chains.192
Analyzing the structural geometry, they suggested that Asp221 is
likely to be protonated and is in position to act as the catalytic
acid.192 Mutation of this TrCel6A residue to alanine results in
complete annihilation of activity, where the differences in
activity are not attributable to substrate binding.192,656 This
finding is consistent across the GH6 family including bacterial
and fungal representatives, as well as in EG and CBH functions
(refs 194, 479, 574, 656, 658, 670, and 680−684). Structural
studies have also captured the catalytic acid in two distinct
orientations.194,656,659,670,683,685 While the two conformations
have been attributed to different protonation states, Varrot et al.
suggest the conformations may represent accommodations
made within the active site to enable procession of the cello-
oligomer.665
7.2.2. pKa Modifying Residues. In addition to the catalytic
acid, it is generally accepted that the GH6 catalytic mechanism
relies on an adjacent secondary aspartic acid that serves, at
minimum, to prime the catalytic acid pKa. Upon solution of the
first GH6 structure, Rouvinen et al. posited that Asp175 could
force protonation of Asp221 and stabilize a transitional positive
charge at the substrate ring oxygen −1 binding subsite.192 Site-
directed mutagenesis and characterization of activity confirmed
the D175A mutant displayed approximately 20% of the activity
of wild-type, which suggests the residue plays a supporting role
in catalysis.192 The putative role of Asp175 as a pKa modifying
residue was again suggested by Koivula et al., who observed
significantly reduced, but measurable, activity of the D175A
mutant.656,684
Similar behavior has been observed in bacterial GH6
representatives as well. Studies characterizing the role of the
homologous C. f imi Cel6A residue, Asp216, again suggested an
indirect role of the residue in catalysis. As a result of the D216A
mutation, Damude et al. observed reductions in activity of 18- to
1380-fold depending on the substrate, but they noted the
mutants maintained a considerable amount of activity.661 The
homologous residue in Tf Cel6A (Asp79) is also reported to
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function as a pKa modifying residue despite its structurally
observed location 11 Å away from the catalytic acid.680
Wolfgang et al. obtained pH activity profiles of the D79A,
D79E, and D79N mutants demonstrating that Asp79 raises the
pKa of the corresponding catalytic acid.
Besides modulating pKa and potentially acting as the catalytic
base, as discussed in the next subsection, additional roles for
Asp175 and its homologous residues have been suggested.
Koivula et al. noted that Asp175 is ideally positioned to function
in stabilization of the oxocarbenium-like transition state.574 In a
later study, the authors used molecular modeling and structural
studies with fluoroglycosyl ligands to investigate this hypoth-
esis.656 They confirmed Asp175 stabilizes the exceptionally
electron-deficient fluorine transition state and suggest this
interaction also takes place with oligosaccharides. Interestingly,
Vuong et al. found that the D226A Tf Cel6B mutant,
homologous to Asp175, could hydrolyze a soluble substrate at
the same level as wild-type producing large oligosaccharides, but
the same mutant could not hydrolyze crystalline cellulose to
produce cellobiose. This latter finding suggests this residue may
play a role in processivity.682 Tamura et al. also suggest this
residue plays a crucial role in active site loop functionality in
CcCel6A and CcCel6C, as deletion enabled a significant
conformational change (Figure 50).671
Asp263 in TrCel6A and its homologous residues have also
been suggested to serve as pKa modifying residues. Structurally,
this aspartic acid sandwiches the catalytic acid with an adjacent
aspartic acid. Originally, Rouvinen et al. suggested that Asp263,
or alternatively Asp401, may act as the catalytic base.192 Later,
several studies successfully ruled out Asp236 as the catalytic base
and suggested Asp263 likely increases the pKa of the catalytic
acid.194,659,661 Site-directed mutagenesis and activity measure-
ments of the homologous residue (Asp156) in Tf Cel6A support
this proposal, with decreased activity found for D156A and
D156N but not for D156E.680
7.2.3. Catalytic Base. The definitive assignment of a
catalytic base in the inverting GH6 mechanism remains an open
question. Recent evidence even suggests that GH6 enzymes do
not require a direct catalytic base as part of their single-
displacement hydrolytic mechanism (i.e., a residue that could
accept a proton directly from the attacking nucleophilic water
molecule).
The structural study of TrCel6A by Rouvinen et al. was one of
the first instances in which Asp401 was proposed as the catalytic
base.192 Spezio et al. later proposed that the homologous
residue, Asp265, in Tf Cel6A would aid in activity by ensuring
the enzyme’s charged state, effectively as the catalytic base.658
Damude et al. later used kinetic measurements to investigate the
role of this residue, Asp392, in Cf Cel6A.661 After the catalytic
acid mutant, the Cf Cel6A D392A mutant displayed the second-
largest reduction in activity with a decrease in activity of
approximately 3 × 104 on both CMC and PASC. Combined
with geometric considerations from the crystal structures of
TrCel6A and Tf Cel6A, the authors posited that Asp392, Asp401
in TrCel6A, serves as the catalytic base.
The Damude et al. study has been the source of much
confusion over the years, as only a handful of other studies
suggest this residue causes such a drastic decrease in catalytic
activity. One of the first suggestions that TrCel6A’s Asp401 may
not actually be the catalytic base was put forth by Koivula et al.,
who noted that the salt bridge in which Asp401 participates
would likely interfere with proton abstraction from the attacking
water molecule.574 The presence of water molecules near the
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catalytic center led some researchers to briefly consider the
possibility that catalysis proceeds via Grotthuss mechanism in
GH6s.170,659 However, the prospect was ultimately dismissed
(and then later re-examined) on the basis of the Damude et al.
study identifying the Asp401 equivalent as a catalytic base.
In a comprehensive examination of the aspartic acids of
Tf Cel6A, Wolfgang et al. determined that various Asp265
mutants (homologous to TrCel6A Asp401) retained anywhere
from 2% to 10% of wild-type activity on CMC and PASC.680
This suggested that the residue was not playing a direct role in
catalysis, such as a catalytic base would. To test whether Asp265
and Asp79 (the pKa modifying residue) functioned coopera-
tively as a base providing residual activity when only one was
mutated, the authors tested the activity of the double mutant
D79N/D265N. The observed results were similar to what
would be expected from a cumulative, independent decrease
rather than a marked decrease resulting from removal of the
catalytic base.
Molecular modeling studies were particularly insightful in
developing what is our current understanding of how GH6
enzymes proceed without a traditional catalytic base. Modeling
the TrCel6A transition state as an oxocarbenium cation, Koivula
et al. scanned several initial configurations of the active site to
identify potential catalytic base residues. However, the authors
concluded there were no suitable candidates to act as a direct
base. Some of the models revealed conformations wherein water
molecules were positioned so as to attack the anomeric carbon
of the oxocarbenium cation. The Ser181 side chain and the
Asp401 backbone stabilized one water molecule, which also
connected to the Asp175 side chain via a second water molecule.
After bond cleavage in the simulation, the distorted −1 sugar
ring relaxed to the chair conformation and moved slightly out of
the active site. Koivula et al. thus suggested there is little need for
a protein-based catalytic base and instead suggested a proton
could be transferred from the nucleophilic water to Asp175 by
means of a second water in a Grotthuss-type mechanism; thus,
Asp175 could act as an indirect catalytic base via a second water
molecule. In addition to reviving the notion of a water-mediated
Grotthuss mechanism, the authors also predicted the 2,5B
conformation of the oxocarbenium cation intermediate.
Compounding structural studies capturing transition state
intermediates would later confirm this for other GH6
enzymes.665,666 To date, the assignment of Asp175 as the
catalytic base is the most plausible hypothesis that has been put
forth.
As definitive evidence of the role of Asp175 remains open, the
search for a suitable GH6 catalytic base continues. In Tf Cel6B,
Vuong et al. mutated all potential catalytic base residues within 6
Å of the −1/+1 subsites to an alanine.682 The authors suggested
that an exogenous nucleophile such as sodium azide could
partially “rescue” enzyme activity in the absence of a catalytic
base and thus tested this hypothesis. Sodium azide did not
“rescue” activity for any of the mutants nor was a catalytic base
identified. However, the catalytic relevance of each of the
mutated residues was confirmed. Activity of the D226A/S232A
(equivalent to TrCel6A Asp175 and Ser181) double mutation
was partially “rescued” by low concentrations of sodium azide.
The authors suggest that the azide ion activates a water molecule
that performs hydrolysis. This “rescue” suggests Asp226 and
Ser232 activate a catalytic water via a proton-transferring
network as a means of hydrolysis. The Tf Cel6B structure of the
D226A/S232A double mutant later supported this hypoth-
esis.686
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7.2.4. Catalytic Priming of Ring Distortion. While many
residues are likely involved in the catalytic priming of the
substrate for catalysis, the centrally located tyrosine has been
shown to function in steric distortion of the −1 glucose
moiety.574 An early investigation by Koivula et al. effectively
established the role of Tyr169 and homologous tyrosines
through crystallography and biochemical characterization of
Tyr169 mutant activity.574 Through structural evidence and
characterization of pH dependence of cellotetraose hydrolysis in
the wild-type and Y169F variant, the authors proposed that
Tyr169 contributes to ring distortion in the −1 subsite as well as
helps to ensure protonation of the catalytic acid.574 Zou et al.
later confirmed Tyr169 aids in distortion of the −1 glucose but
noted that the Y169F mutant exhibits the same distortion.194
Later, molecular modeling studies confirmed the strongly
stabilizing nature of Tyr169 in maintaining the distorted 2SO
intermediate conformation.656
Similar results in homologous GH6s suggest the tyrosine at
the −1 binding subsite has conserved function. In the bacterial
Tf Cel6A, Tyr73 mutants display tighter binding and lower
hydrolytic activity in comparison to wild-type.687,688 Barr et al.
proposed that the additional volume created by these mutations
allows the −1 subsite sugar ring to bind in the relaxed 4C1
conformation, indicating the role of Tyr73 could be to align the
−1 sugar into an optimal position relative to the catalytic
residues.687 Alternately, they noted that Tyr73 could stabilize an
intermediate oxocarbenium ion through cation−π interaction,
consistent with the observation that Y73S had lower activity
than Y73F.687,689 In Tf Cel6A, Tyr73 is further from the catalytic
acid than homologous residues in TrCel6A, and thus would only
be able to aid in pKa modulation through conformational
changes of the active site. The same roles have been reported for
the −1 binding site tyrosines of HiCel6A and Tf Cel6B as
well.666,682686
7.2.5. Substrate Binding. As with most GHs, family 6
cellulases exhibit aromatic-lined substrate binding sites in both
cleft and tunnel architectures. The binding sites have been
historically defined by the number of glucose moieties that may
bind along the length of the tunnel or cleft. Since GH6 enzymes
are nonreducing end specific, positive numbers denote the
“substrate” side of the active site, and negative numbers denote
the “product” side. The only known exception to this
generalization appears to be the GH6 CBH from T. emersonii
that has been reported to act from both the reducing and
nonreducing end of cellulose.513 TrCel6A exhibits at least six
binding subsites from +4 to −2.525,606 Four of these sites, +2 to
−2, were observed in the seminal TrCel6A structure and have
been extensively characterized over the years.192,574,690−693 The
overall number of binding sites across GH6s varies among
members from at least 4 and up to 8 depending on the
enzyme.574,665,667,694,695 However, generalizations as to the roles
of residues in the core binding sites +2 to −2 are insightful and
will be discussed here.
Product side binding sites, −2 and −1, have been shown to
tightly bind the ligand aiding in both positioning the glycosidic
linkage for catalytic cleavage and stabilization of the cleaved
dimeric product. In TrCel6A, the −2 subsite exhibits a
carbohydrate−aromatic stacking interaction mediated by a
tryptophan residue, Trp135. Little experimental evidence
characterizing this binding site in TrCel6A is available; however,
molecular modeling studies suggest the tryptophan residue
tightly binds the ligand to maintain both the −1 ring distortion
and for product stability.578 This strong association of the
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product with the −1 and −2 subsites may in fact contribute to
the moderate product inhibition observed in
TrCel6A.344,581,599,606,693 Studies of bacterial GH6s Tf Cel6A
and Tf Cel6B have been influential in defining protein−substrate
interactions in the −2 binding site.687,696 In Tf Cel6A, site-
directed mutagenesis of a lysine residue to a histidine in the −2
binding site was shown to improve performance on PASC over
filter paper, suggesting this residue plays a rate-limiting role in
hydrolysis of crystalline cellulose.696 In examining Tf Cel6B,
Vuong and Wilson identified an asparagine, Asn282, which
significantly impacts the ability of the enzyme to hydrolyze
crystalline substrates. Mutations of this asparagine to alanine
and aspartate to alanine increased overall activity on BMCC and
filter paper and increased processivity.528 Overall, character-
ization studies suggest the −2 binding site is important for
catalysis, processivity, and product binding.
Distortion of the −1 glucopyranose ring in the active sites of
GHs is thought to be necessary for catalysis, as discussed
previously. In GH6s, this distortion occurs on the product side
of the cleavage site. A number of studies focus on understanding
both how enzyme structure contributes to distortion and
energetic contributions from distortion. Study of the first
cellulase structure, TrCel6A,192 as well as several subsequent
structural studies, observed tilting of the −1 glucopyranose ring
out of the plane of the other substrate units and noted puckering
of the ring from the relaxed chair conforma-
tion.194,209,574,665,666,683 Early molecular modeling simulations
of Tf Cel6A in complex with cellotetraose focused on
investigating the conformation of the glucosyl unit bound in
the −1 subsite confirming the stability of the −1 puckering.692
Koivula et al. noted that while TrCel6A subsites −2, +1, and +2
contain tryptophan residues, the −1 subsite is instead composed
of a lysine, aspartate, and a tyrosine (described above), all of
which likely contribute to the distortion of the sugar.574 The
authors also proposed that binding in the +2 subsite causes
strain that manifests as ring distortion in the −1 subunit based
on the 700-fold higher specificity constant for cellotetraose over
cellotriose. Consistent with the proposal that interactions
beyond the −1 subsite are involved in ring distortion and
productive substrate binding,574,690 Payne et al. used free energy
calculations to find that mutations to tryptophan residues in
multiple binding sites affected the conformation of the −1
sugar.578 Specifically, mutating the +2, +1, and −2 tryptophans
(Trp269, Trp397, and Trp135, respectively) to alanine resulted
in the −1 sugar ring relaxing to a 4C1 conformation. Mutation of
the +4 site tryptophan, Trp272, did not cause ring relaxation,
however. Using molecular simulation, Bu et al. further revealed
that the relative stability of the distorted conformation is
sensitive to pH, with the 2SO conformation favorable at the
TrCel6A optimum pH of 5.479
Exomode initiation in processive enzymes is thought to occur
via the acquisition of free chain ends, which are threaded into
the active site. Accordingly, the enzymes appear to exhibit
machinery enabling acquisition through carbohydrate-π stacking
with an aromatic residue at the entrance of the enzyme active
site. Koivula et al. hypothesized that TrCel6A exhibited an
additional two binding sites, +3 and +4, wherein Trp272 in the
+4 binding site plays a key role in chain acquisition.525 Trp272
mutants retained near wild-type hydrolytic capability on
amorphous cellulose but were significantly impaired on
BMCC. At the same time, the binding affinity of the mutants
was similar to wild-type, confirming the hypothesis that the
residue was critical to chain acquisition. Notably, this
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Figure 51. Hypothesized processive catalytic cycle for exo-initiated attack. Pre-slide mode: The “more open” TrCel6A structure, 1QK2, represents the
“pre-slide” mode conformation of a processive GH6.194 Loop A (pink cartoon) is in the open conformation with Ser181 far from the catalytic center.
The catalytic acid Asp221 is hydrogen bonding with Asp175, the pKa modifying residue and putative catalytic base. Residues thought to participate in
catalysis, directly or indirectly, are shown in green stick. Tf Cel6B was captured in a similar “more open” conformation with a full-length cello-oligomer
in the 4AVO structure.686 Thus, we use the 4AVO cello-oligomer (cyan stick) in the pre-slide mode, slide mode, and Michaelis complex panels. Slide
mode: The cello-oligomer processes through the active site, filling the −1 and −2 binding sites. The protein (1QK2) has not yet changed
conformation in slide mode allowing the oligomer to pass unobstructed. Michaelis complex: The protein undergoes a conformational change
positioning loop A near the catalytic center, represented here by the TrCel6A 1QJW structure.194 In the Michaelis complex, the backbone of Asp401
and the side chains of Asp175 and Ser181 form a network mediated by two water molecules, red spheres. The catalytic residues illustrating the
Michaelis complex have been selected from various structures to represent this intermediate state (1QJW, Asp175, Asp401, and Ser181, water
molecules; 1QK2, Tyr169; 1HGW, Asp221 from chain B).656 Substrate−product complex: Hydrolysis occurs, putatively via the Grotthuss
mechanism, breaking the glycosidic linkage. The protein (1QJW) maintains the tightened active site conformation throughout hydrolysis producing
an α-cellobiose product molecule. The product and substrate ligand shown is that of the Tf Cel6B 4AVN structure with a modeled −1 glucose based
on the 4AVO ligand.686 The product molecule is expelled, and the processive catalytic cycle is reset.

tryptophan is conserved in GH6 CBHs but not in EGs. Zhang et
al. later confirmed the Tf Cel6B tyrosine functions in a similar
manner.526
7.2.6. Product Inhibition. GH6 product inhibition has not
been as extensively examined as in complementary GH7
cellulases (see section 6.3). Several early studies examined T.
reesei cellulase product inhibition, using cocktails rather than
purified enzymes.581,599 These studies reached the conclusion
that cellulases in the cocktail were indeed inhibited by the
cellobiose product, but it became clear this effect was likely
dominated by the interaction of TrCel7A with cellobiose rather
than TrCel6A. At 25 °C, TrCel6A has a reported cellobiose
inhibition constant (Ki) of approximately 500 M−1,301 while the
inhibition constant for TrCel7A with cellobiose is roughly
50 000 M−1.607 Following on from this work, Teleman et al.
developed a hydrolysis progress curve model including product
inhibition parameters and went on to suggest the TrCel6A
cellobiose inhibition may be closer to 5000 M−1 than 500
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M−1.691 Nevertheless, cellobiose inhibition in TrCel6A pales in
comparison to values observed for TrCel7A. Moderate levels of
glucose inhibition of TrCel6A have also been observed with an
inhibition constant of 150 M−1.301,691 The inhibition of TrCel6A
has been described as noncompetitive inhibition and can reduce
cellotriose hydrolysis by a factor of 30.691 However, glucose
inhibition does not appear to be universal across GH6s, as
glucose did not significantly inhibit Tf Cel6A hydrolysis of
CMC697 or Avicel.698
Attempts to understand molecular contributions to cellobiose
product inhibition in GH6s are accordingly limited. Molecular
simulation suggested that the strong substrate binding at the −2
subsite likely contributes to TrCel6A product inhibition.578 Site-
directed mutagenesis of Tf Cel6B active site residues has been
shown to alleviate cellobiose inhibition.526 However, this
improvement in hydrolysis comes at the price of ability to
degrade crystalline cellulose. In the same study, a glycine to
proline loop mutation inexplicably increased activity on
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amorphous and crystalline substrates while increasing cellobiose
inhibition. A molecular-level explanation of this observation
remains elusive.
7.2.7. Processive Catalytic Cycle. The inverting catalytic
mechanism described here is common to all GH6 enzymes.
CBHs in the GH6 family are also thought to exhibit an
encompassing “processive catalytic cycle” wherein the hydrolysis
step is one part of the entire process of successively cleaving
crystalline polysaccharides (see section 6.2). In this section, we
outline the steps of the hypothesized processive catalytic cycle.
We note that this is a hypothesized mechanism and that some of
the steps, particularly “pre-slide mode” and “slide mode”, are not
likely accurate indictions of endo-initiated attack by GH6s. The
cycle presented here includes the original proposal by Zou et al.,
described above, and combines additional structural-based
evidence from processive CBHs that followed the Zou et al.
study.194,573 Figure 51 illustrates the hypothesized processive
catalytic cycle using structures available in the literature. The
putative steps include the following: (1) In the pre-slide mode,
the processive catalytic cycle begins after the GH6 has acquired
a free chain end from the nonreducing end of the polysaccharide
as part of the initial processivity event.20 The ligand occupies the
substrate binding sites (+1 through +4 or beyond, depending on
the enzyme), while the product binding sites (−1/−2) remain
unoccupied. At this point, the active site loops are in the “open”
conformation such that the side chain of the serine implicated in
catalysis is more than 10 Å away from the catalytic center. The
catalytic acid (Asp221) and pKa modifying/putative base
aspartate (Asp175) hydrogen bond with each other. (2) In
the slide mode, the cello-oligosaccharide proceeds through the
active site tunnel by a cellobiose unit filling the −1 and −2
binding sites. The −1 glucose moiety becomes distorted
through steric interactions with a tyrosine. Asp221 is protonated
during this step but maintains its interaction with Asp175.
Interaction of this latter aspartate with a neighboring arginine
allows it to maintain the charged state. (3) In the Michaelis
complex, a conformational change in loop A breaks the
interaction of the catalytic acid with Asp175. As a result, the
protonated catalytic acid rotates toward the glycosidic linkage.
The active site tunnel closes, and the serine residue of loop A
(Ser181) moves toward the catalytic center. Two water
molecules are stabilized by newly formed interactions with
serine and two aspartates (Asp175, Asp401). (4) In the
substrate−product complex, hydrolysis occurs via the Grotthuss
mechanism leaving an inverted α-cellobiose product in the −1
and −2 subsites. The product is expelled from the active site,
potentially with a conformational change of loop A, and the
processive catalytic cycle begins anew.
7.2.8. Synergistic and Processive Function. Efficient
polysaccharide deconstruction has long been known to require a
complex mixture of CBHs, a host of EGs, and accessory
enzymes all working together through complementary func-
tion.32 Synergistic activity between TrCel6A and TrCel7A was
reported as early as 1980 prompting an entire body of research
devoted to understanding the mechanisms behind this
phenomenon.436 Chanzy and Henrissat hypothesized that
orthogonal action of TrCel6A and TrCel7A could be
responsible for their synergy.302 As discussed previously, it
eventually became apparent that GHs do not exist as strictly
delineated “endo” and “exo” active enzymes suggesting
additional possibilities for synergistic function of the two
enzymes.304 Ståhlberg et al. suggested T. reesei CBHs are capable
of two essential modes of cellulolytic degradation.304 In line with
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its classification as a CBH, TrCel6A may acquire a free chain end
and processively cleave hydrolytic linkages. However, on the
basis of production of reducing ends on both PASC and filter
paper as well as reversible binding, TrCel6A was reported to
exhibit a high degree of endo-initiation activity allowing the
enzyme to randomly hydrolyze internal glycosidic bonds. In
TrCel6A, this ability to cleave internal linkages similar to that of
an EG has been attributed in part to the flexibility of the tunnel-
forming active site loops.304 Later, Harjunpää would report that
TrCel6A exhibits strictly processive action, finding no
cellotetraose yield from cellohexaose, as would be expected if
the enzyme randomly cleaved glycosidic linkages.693 These
opposing findings hint at the difficulty in describing exo-activity
and processive function using accessible characterization
techniques. Currently, no consensus approach has been widely
adopted in examination of GH6 exo-activity, and thus,
comparison of findings across laboratories is difficult at best.
Difficulties associated with cellulase processivity measurements
have been described above in section 6.2, and a recent review
outlines limitations of available techniques, which are currently
substantial.521 With this in mind, we describe our evolving
understanding of the mechanism by which GH6s deconstruct
cellulose, controversy in description of processive function, and
technologies being developed to move forward in our
understanding of GH6 processive function.
Description of the endo- or exo-initiation character of GH6
cellulases has long been qualitatively determined through
soluble reducing sugar assays, where enzymes exhibiting a
greater degree of exo-initiation activity produce fewer insoluble
sugars relative to more endo-active enzymes.523 According to
this approach, Tf Cel6A exhibits a high degree of endo-activity
relative to the high exo-active character of Tf Cel6B. In this same
study, TrCel6A and Tf Cel6B were reported to be functionally
equivalent in synergistic mixtures of cellulases indicating a
similar mechanism in degradation of cellulose. The conclusion
that TrCel6A is an exo-active cellulase was long unquestioned.
However, recent characterization and visualization studies now
suggest GH6 CBHs may also exhibit strongly endo-character-
istic activities304,476,546,600,699,700
Several subsequent visualization studies support the hypoth-
esis that CBHs TrCel6A and HiCel6A exhibit a high degree of
endo-initiation. TEM captured clear images of the effects of
incubating HiCel6A, HiCel7A, and a mixture of the two on
digestion of BC ribbons.540 Upon incubation with HiCel7A,
Boisset et al. observed thinning of the cellulose ribbons
indicative of processive degradation of the polysaccharide.
When the BC was incubated with HiCel6A, the fibrils were also
thinned in several locations, but more importantly, the fibrils
were cut into shorter fragments. The latter behavior is suggestive
of endo-initiated degradation of the cellulose fibrils. Together,
HiCel6A and HiCel7A exhibit exo/exo synergy, working from
opposite ends of the polysaccharide and using complementary
modes of degradation. Boisset et al. illustrated this phenomenon
in a schematic reproduced here in Figure 52.540 The authors
made the important observation that choice of substrate greatly
affects the outcome of the experiment. They argued that the BC
ribbons are the optimal substrate for observing the endo-
processive action of HiCel6A; this substrate does not exhibit the
abnormally high degree of structural defects of pretreated
substrates such as BMCC, Avicel, or even Valonia microfibrils
that may preclude endo-activity.
Recently, real-time visualization of cellulase action on
cellulose using high-speed AFM has been reported.476 Igarashi
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Chemical Reviews
Figure 52. Schematic of the action of HiCel7A and HiCel6A on BC
ribbons. The two enzymes exhibit complementary function, working
from the reducing end, R, and the nonreducing end, NR, of the
microfibril, respectively. (A) The primarily exo-acting HiCel7A
sharpens the microfibril into long, thinner ribbons. (B) HiCel6A
exhibits a dual mode of action, thinning the ribbons in certain regions
but also cutting the fiber using endo-initiated hydrolysis. Reprinted with
permission from ref 540. Copyright 2000 American Society for
Microbiology.
et al. investigated the synergistic effects of TrCel7A and
TrCel6A finding, as has often been reported, that the two act in
a synergistic exo/exo fashion. Interestingly, the authors noted
that when an ammonia pretreated cellulose polymorph,
cellulose IIII, was incubated with only TrCel6A for 3 and 8
min, little change in morphology of the microfibril was
observed.476 However, subsequent addition of TrCel7A resulted
in remarkably faster digestion than with either enzyme alone. It
seems reasonable to conclude that TrCel6A may function in a
highly endo-active mode on cellulose IIII creating new chain
ends for TrCel7A that would otherwise be unavailable. It has
also been speculated that TrCel6A may even synergistically
remove obstacles from the path of TrCel7A resulting in
enhanced hydrolysis. One of the more intriguing results of
this work relative to TrCel6A can be seen in the movies
supporting the manuscript; in isolation, TrCel6A appears to be
nonprocessive. Over the time span of the high-speed AFM
measurements, the enzyme molecules do not deviate signifi-
cantly from their initial positions. This latter finding is counter
to the community’s working hypothesis that TrCel6A is a
processive cellulase and highlights the need for accurate GH6
processivity assays.
Much of the difficulty in uniformly characterizing GH6
activity, degree of processivity, and initial mode of attack arises
from the fact that there are relatively few model substrates
available for nonreducing end specific enzymes. For decades,
reducing end specific assays have taken advantage of
chromogenic and fluorogenic cellobiosides and lacto-
sides.701−703 The specific chemistry of the reducing end of
cellulose and cello-oligomers allows for attachment of reporter
molecules, whereas the nonreducing end lacks this unique
1379
Review
chemistry.521 Use of reducing-end attached chromogenic or
fluorogenic molecules in examining GH6 activity is generally
uninformative, as the enzymes exhibit relatively poor hydrolysis
of the heteroglycosidic linkages or preferentially cleave the
homoglycosidic linkage of longer model substrates. A recent
advance in fluorogenic substrates for characterization of GH6
activity made use of molecular docking studies to understand
how GH6s bind 4-methylumbelliferyl-β-D-cellobioside sub-
strates and suggested rational improvements based on the
findings.704 Wu et al. discovered that GH6 enzymes may
nonproductively bind with the traditional umbelliferyl substrate
and that substitutions at the C4 or C6 position of the
umbelliferyl motif greatly improved hydrolytic turnover of the
heteroglycosidic linkage.704 The 6-chloro-4-methyl-umbelliferyl-
β-D-cellobiose reporter molecule was deemed to be the most
successful improvement, as the molecule demonstrated an
appropriate balance of activity, solubility, and fluorescence in
comparison with other C4/C6 modifications. Nevertheless,
GH6 turnover of these molecules remains low when compared
to GH7 activity. The authors optimistically interpreted this to
mean there are additional gains to be had in design of GH6
reporter molecules.
7.3. Glycosylation
To our knowledge, TrCel6A is the only GH6 for which
glycosylation has been explicitly characterized. Hui et al. used a
combination of capillary liquid chromatography-electrospray
and matrix-assisted laser desorption and ionization time-of-flight
mass spectrometry to locate N-linked glycans on the CD and
quantify the number of O-linked glycans attached to the CBM
and linker domain.418 The TrCel6A enzyme was purified from
the T. reesei RUT-C30 strain fermentation broth and thus
closely reflects the native glycan arrangement. The cleaved
CBM-linker domain, from residues 1 to 82, exhibits between 39
and 46 O-linked mannose residues appended to the threonines
and serines in this region. This is significantly higher than the
number of O-linked glycans observed on the linker of TrCel7A,
related to the shorter GH7 linker. As the linker length across
GH families appears to be conserved,417 this finding may be true
of other fungal GH6 enzymes exhibiting linker regions. On the
CD of TrCel6A, Hui et al. report there are three N-linked glycan
sites at Asn14, Asn289, and Asn310. Of these three, the Asn310
glycan was reported to be a high-mannose glycan with anywhere
from 7 to 9 mannose residues attached to the base GlcNAc2.418
This finding was in line with observations made in crystallization
of the first TrCel6A structure. Rouvinen et al. found that the
TrCel6A CD likely exhibited N-linked glycans at the Asn289
and Asn310 residues, though the only assignment made was a
single GlcNAc at Asn310.192 The crystal structure also reported
the CD as having O-linked glycans at residues Thr87, Thr97,
Ser106, Ser109, Ser110, and Ser115. Additional crystal
structures of the natively expressed TrCel6A CD also exhibit
various combinations of the attached glycans.194,656,704
Insights into glycosylation of GH6 cellulases other than
TrCel6A are limited to findings from structural reports.
Unfortunately, the C. cinerea and C. thermophilum GH6
structures were obtained through recombinant expression in
E. coli; thus, the structures do not exhibit glycosylation.670−672
The HiCel6A structures were expressed in an alternative fungal
host, A. oryzae, and do exhibit some glycosylation captured in
the structures. Varrot et al. reported the CD exhibits an N-linked
GlcNAc at Asn141 and at least two O-linked mannose residues
at Ser127 and Thr118.659 It is likely the CD may exhibit more or
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Chemical Reviews
rather different glycans on the surface of the CD in the native
host, but the extent to which this is true is currently publicly
unavailable.
7.4. Protein Engineering
As with their GH7 counterparts, engineering GH6 CBHs
toward higher thermal stability has received significant attention
from industrial and academic laboratories. Early efforts focused
on improving GH6 stability were targeted toward bacterial
representatives. Zhang et al. attempted to improve Tf Cel6B
thermostability by introducing disulfide bonds.526 They created
a Tf Cel6B G234S-G284P double mutant that showed 2-fold
increase on filter paper, but the benefit did not carry over to
synergistic mixtures.526 Ai and Wilson attempted to engineer a
more thermostable Tf Cel6B by introducing a disulfide bond by
means of the N233C-D506C double mutant (residues Asn233
and Asp506 in Tf Cel6A correspond to Asn182 and Arg410 in
TrCel6A).705 The circular dichroism spectra of the wild-type
and double mutant were identical, indicating no change in the
final structure. The mutated residues joined the two loops
covering the active-site cleft but did not inhibit activity on CMC
or PASC. Moderate gains in thermal stability were attained
through the double mutation with 100% of activity maintained
at 50 °C for 20 h compared to 85% activity retention in the wild-
type.705 Unfortunately, the mutations caused a decreased
protein yield attributed to lower in vivo expression but
prohibiting extension to commercial applications.706
In efforts to increase thermostability in fungal GH6 cellulases,
Lantz et al. targeted more than 100 nonconserved TrCel6A
residues for mutagenesis. Therein, they concentrated primarily
on the CD surface residues hypothesizing that these residues
benefit the least from hydrophobic packing stability and may
exhibit the largest gains; some linker and CBM sites were also
examined.707 Through this comprehensive approach, gains in
thermal stability of nearly 7 °C for TrCel6A (and 15 °C for
TrCel7A) were achieved. These gains in thermal stability were
significant enough to raise the stability of the primary hydrolytic
components to that of the more thermally stable EGs TrCel5A
and TrCel3A in a commercial enzyme cocktail. An added side
benefit of the thermal stability mutations was a moderate gain in
TrCel6A activity.
Additional efforts to improve GH6 thermal stability include a
significant body of work from the Arnold group, as previously
described in the GH7 section.674,708−710 Heinzelman et al. first
employed structure guided enzyme recombination of HiCel6A,
TrCel6A, and the bacterial GH6 CtCel6A.709 From a sample set
of 48 chimeras secreted by a glycosylation-deficient strain of S.
cerevisiae, five chimeras exhibited half-lives of inactivation (63
°C) higher than the most thermostable wild-type parent
(HiCel6A). None of the three chimeras were more active on
PASC at 50 °C than TrCel6A, the most active parent, but each
were active at higher temperatures than the parents, retaining
activity up to 70 °C. The most thermostable parent, HiCel6A, is
inactive above 57 °C. They named the most active chimera
HJPlus, which was created by substituting the three “blocks”
predicted to be most stabilizing into TrCel6A. In a follow-up
study, Heinzelman et al. produced additional GH6 chimeras that
their regression model predicted would encode thermostable
chimeras.708 The block that most strongly contributed to
thermostability contained 10 differences between two parents
with different thermostabilities. Mutating each residue in the
block identified one mutation, S313C in HiCel6A, which
reduced the T50 (temperature at which an enyme loses 50% of
1380
Review
activity during a 10 min incubation) by approximately 10 °C.
They tested Cys-Ser mutations on parent enzymes and found an
approximately 8 °C increase in T50 for TrCel6A C400S (where
the numbering is for the full-length enzyme with a CBM-linker).
This corresponding Cys to Ser mutations further improved
secretion of the functional enzyme. They proposed that the
increased stability of the Cys-Ser mutation could be due either
to stronger hydrogen bonding interactions or due to steric
reasons, as the Cys residue resides closer to the carbonyl of
Pro339 than Ser.
Wu and Arnold further increased the HJPlus chimera
thermostability via directed evolution, creating the new chimera
3C6P, which contained seven mutations over HJPlus: S30F,
V128A, M135L, Q277L, S317P, S406P, and S413P.674 The
mutations creating the 3C6P variant were tested one-by-one in
TrCel6A. All mutations except Q276L (equivalent to Q277L in
3C6P) either stabilized or did not affect the T50 of the parent
TrCel6A enzyme. The five thermostabilizing mutations in 3C6P
are all near the surface, with the Ser to Pro mutations being the
only solvent exposed mutations in loop regions. Wu and Arnold
proposed that they limit conformational freedom without
straining the backbone (the Cα and Cβ atoms in TrCel6A
and HJPlus align well with the Cα and Cβ of the corresponding
prolines in 3C6P). 3C6P displayed a half-life of 280 min at 75
°C and a T50 of 80.1 °C, a 15 °C increase over HiCel6A and 20
°C increase over TrCel6A. The 3C6P activity at its optimal
temperature of 75 °C offers a 10-fold reduction in hydrolysis
time compared to HiCel6A activity at its optimal temperature of
60 °C on Avicel (at 60 h). Crucially, it continued to demonstrate
synergy, and they found that a mixture of thermostable Cel6A
(3C6P) and a thermostable variant of Cel7A from the same
group650 releases 1.8 times more cellobiose than a wild-type
mixture at their respective optimum temperatures of 70 and 60
°C from Avicel.
On the basis of results from their previous studies wherein
free cysteine residues were found to be detrimental to stability in
GH6 cellulases,708,709 Wu et al. investigated the role of paired
and free cysteine residues in GH6 thermal degradation.710 They
noted that some GH6 enzymes contain unpaired, free cysteine
residues, including the thermostable chimera 3C6P Cys246.
Mutants (C246A, C246G, C246L, and C246S) lacking the free
cysteine showed increased extreme (90 °C) temperature
tolerance. They noted that HiCel6A and TrCel6A have a free
cysteine residue and lower temperature tolerance than wild-type
CtCel6A, which does not have a free cysteine. As with 3C6P,
mutants of HiCel6A and TrCel6A in which their free cysteine
residues were removed showed improved tolerance of high
temperature incubation. Further study of the C246G mutant
indicated that it was able to retain activity after high temperature
incubation due to disulfide-bond-assisted refolding to a
productive conformation. They proposed that GH6 thermal
inactivation is due to disulfide-bond degradation and thiol-
disulfide exchange that results in misfolding, and that removing
free cysteine residues limits degradation by the later mechanism.
GH6 enzymes display a diverse range of pH optimum and
activity ranges (Table 9).192,499,680,711,712 Nevertheless, it is
frequently desirable from an industrial perspective to under-
stand both the molecular contributions to activity at various pHs
and methods for engineering proteins more tolerant of pH
conditions beyond optimal. Several groups have investigated the
effect of substrate binding on the pKa of the enzyme as well as
the effect of pH on active site loops and activity. Damude et al.
noted that Cf Cel6A displays a basic shift in pKa with substrate
DOI: 10.1021/cr500351c
Chem. Rev. 2015, 115, 1308−1448
 Table 9. Summary of Biochemical Characterizations of Fungal GH6 Cellulases
temp opt substrate
organism expression host enzyme pH opt (°C) for opt substrate specificity ref comments
Agaricus bisporus Saccharomyces Cel3 CMC, filter paper, PASC, barley β-glucan Chow et al., 1994714
D649 cerevisiae
Aspergillus nidu- Pichia pastoris X- CBHII 5.5 57 CMC soluble CMC, cello-oligosaccharides Glc3/Glc4/Glc5/Glc6, Bauer et al., 2006487
lans FGSC A4 33
Chemical Reviews

barley β-glucan and lichenan, Avicel

Chaetomium Pichia pastoris Cel6A 4 50 pNPC pNPC, BMCC, filter paper, CMC Wang et al., 2012673
thermophilum
Chrysosporium Cel6A 4.5 60 CMC CMC, labeled CMC, β-glucan Emalfrab et al., 2003715 formerly EG6
lucknowense
669
Coprinopsis cin- Escherichia coli Cel6A 8.0 PASC PASC, Avicel, cellotriose, cellobiose Liu et al., 2009, Tamura et al., assay conditions 30 °C
erea 5338 2012671
Coprinopsis cin- Escherichia coli Cel6B 7.0 PASC PASC, CMC, Avicel Liu et al., 2009 assay conditions 30 °C
erea 5338
Coprinopsis cin- Escherichia coli Cel6C 8.0 PASC PASC, CMC, Avicel Liu et al., 2009 assay conditions 30 °C
erea 5338
540
Humicola inso- Cel6A 60 Avicel Avicel; BC ribbons Boisset et al., 2000, Moriyaet al., assay conditions pH 5.0
lens 2003,716 Wu and Arnold, 2013674
Humicola inso- Saccharomyces Cel6B CMC, PASC, Glc5, reduced Glc6 Schou et al., 1993,442 Dalbøge and assay conditions 30 °C and pH 7
lens cerevisiae Heldt-Hansen, 1994,717 Davies et
al., 2000663
Irpex lacteus Pichia pastoris CBHII 5 50 PASC Avicel, PASC Toda et al., 2008718
MC-2 (Ex-4)
Malbranchea cin- Aspergillus ory- Cel6A PASC, pNPC, Avicel Wu et al., 2011,719 Xu et al., 2009500 assay conditions 50 °C and pH 5.0
namomea zae

1381
Neocallimastix Escherichia coli Cel6A 5.0 40 Avicel Avicel, CMC, PASC, lichenan Denman et al., 1996711 formerly CelA
patriciarum
Neocallimastix Escherichia coli CelA 6.0 50 barley barley β-glucan, lichenan, CMC, Avicel, PASC Wang et al., 2013720
patriciarum β-glucan
J11
Orpinomyces sp. Escherichia coli CelA 4.3−6.8 50 CMC CMC, Avicel, PASC, lichenan, barley β-glucan, pullulan, Glc4 Li et al., 1997712
PC-2
Orpinomyces sp. Escherichia coli CelC 4.6−7.0 40 CMC CMC, Avicel, PASC, lichenan, barley β-glucan, arabinogalac- Li et al., 1997712
PC-2 tan, araban, galactan, pullulan, gum arabic, pachyman,
pustulan, cellotetraose
Orpinomyces sp. Escherichia coli CelF 5.8−6.2 40−50 CMC CMC, PASC, barley β-glucan, lichenan, Avicel Chen et al., 2003721
PC-2
Penicillium de- Cel6A 5.0 50 pNPC pNPC, barley β-glucan Gao et al., 2011722
cumbens JU-
A10
Phialophora sp. Pichia pastoris EgGH6A 7.0 65 CMC-Na CMC-Na, barley β-glucan, Avicel, filter paper Zhao et al., 2012723 retained >40% activity at pH 4.0−10
G5
Piromyces rhizin- Escherichia coli Cel6A 6.0 37−45 CMC CMC, barley β-glucan, lichenan, xylan Tsai et al., 2003724
f latus 2301
Piromyces sp. E2 Escherichia coli Cel6A Avicel Harhangi et al., 2003725 assay conditions 39 °C and pH 6
Podospora anser- Pichia pastoris Cel6A 7 45 Avicel Avicel, CMC, Glc3/Glc4/Glc5/Glc6 Poidevin et al., 2013326
ina S mat+
Podospora anser- Pichia pastoris Cel6B 7 35 Avicel Avicel, CMC, Glc3/Glc4/Glc5/Glc6 Poidevin et al., 2013326
ina S mat+
Podospora anser- Pichia pastoris Cel6C 6 25 Avicel Avicel, CMC, Glc3/Glc4/Glc5/Glc6 Poidevin et al., 2013326
ina S mat+
Review

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Chemical Reviews Review

present, shifting from 5.9 to 6.7 for the protonated group, and
5.7 to 6.3 for the deprotonated group. This shift was attributed

assay conditions 50 °C and pH 5.0

assay conditions 45 °C and pH 5.0

to the substrate excluding water from the active site.695 Recently,
Bu et al. used molecular modeling to gain insights into the
effects of pH on GH6 protein structure and function. Molecular
comments

modeling confirms, as proposed in the Damude et al. study, that

substrate binding results in a basic shift.479 Bu et al. further
demonstrated that pH affects the substrate ring conformation in
the −2 subsite and active site loop flexibility, with more
flexibility found at the optimal pH.
Engineering proteins for pHs beyond the optimum requires
consideration of both the overall protein stability as well as the
Poidevin et al., 2013,326 Ståhlberg et

catalytic function. A particularly successful pH engineering effort

by Wohlfahrt et al. demonstrates that TrCel6A can be altered so
as to maintain both stability and wild-type-like levels of activity
Yamanobe et al., 2000726

at significantly increased pH, similar to what would be

Brown et al., 2007727
ref

Song et al., 2010520

encountered under basic lignocellulosic pretreatment strat-

Wu et al., 2011719

egies.713 The authors targeted three carboxyl-carboxylate pairs

al., 1993304

in a stable arrangement with low solvent accessibility yet near

the flexible active site loops. Under alkaline conditions, the
carboxylic acid group of these pairs would likely be
deprotonated leading to repulsion of the side chain from the
neighboring carboxylate. Mutants were designed replacing the
carboxylic acid with an amide partner. Circular dichroism and
tryptophan fluorescence confirmed the strategy effectively
improved protein stability under alkaline conditions relative to
wild-type. The triple mutant (all three carboxylic acids replaced
Avicel, CMC, Glc3/Glc4/Glc5/Glc6, PASC

by amides) was the most stable of the proteins examined. The

substrate specificity

half-life of this protein was extended by 4-fold at pH 8, and

activity on cellotetraose was unperturbed. Interestingly, the
activity on BMCC was shown to be the same or higher than
wild-type under alkaline conditions. Overall, this protein
engineering strategy appears to represent a general approach
to modification of pH optimums. Furthermore, extrapolation of
this approach toward reducing pH optimum appears straightfor-
ward, namely by substituting amide-carboxylate pairs with
CMC-Na

carboxyl-carboxylate pairs.
PASC

Avicel

PASC

7.5. Conclusions
GH6 cellulases pose a unique challenge in characterization of
substrate

CMC-Na
for opt

molecular mechanisms as a result of their catalytic mechanism

Avicel

and specificity. As such, our molecular-level understanding of

cellulases in this family is somewhat limited in comparison to
other fungal cellulases such as GH7 and GH5 enzymes. The
temp opt
(°C)

following findings summarize the general consensus regarding

45

70

features of GH6 cellulases:

(1) GH6 cellulases employ a one-step, inverting mechanism
pH opt

to hydrolyze glycosidic linkages.

5.0

(2) The GH6 catalytic base has not been definitively

5

identified. Cellulases of this family most likely undertake

hydrolysis via a water-wire mediated Grotthuss mecha-
enzyme

CBHII

CBHII
Cel6A

Cel6A

Cel6A

nism in which a water wire shuttles the resulting proton

from the nucleophilic water molecule to a neighboring
aspartate, namely Asp175 in TrCel6A.
expression host
Aspergillus ory-

Pichia pastoris

Saccharomyces

(3) The GH6 family consists of both CBHs and EGs, both of
cerevisiae

which are capable of endo-initiated attack with specificity

Table 9. continued

toward the nonreducing end of crystalline cellulose.

zae

(4) GH6 CBHs exhibit disordered loops forming the

characteristic tunnel-shaped architecture. The loops
Stilbella annulata

Trichoderma ree-

Trichoderma vir-
Thielavia terrest-
lulolyticus CF-
Talaromyces cel-

demonstrate a remarkable range of flexibility in response

sei QM9414
organism

ide CICC

to both mutagenesis and ligand binding and may be

13038
2612

related to the mechanism of processivity or the endo-

ris

active nature of GH6 CBHs. These loops may also play an

1382 DOI: 10.1021/cr500351c
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Chemical Reviews
indirect role in stabilizing a water molecule as part of the
catalytic mechanism.
(5) GH6 cellulases serve a critical role in the synergistic
activity of cellulase cocktails. They are the only known
nonreducing end specific cellulases and have been shown
to both create new free chain ends for GH7s as well as to
remove obstacles from the path of more processive
CBHs.
(6) While ensuring uniform characterization of GH activity,
degree of processivity, and initial mode of attack is an
inherently difficult task, GH6s pose additional challenges
as nonreducing end specific GHs. Attachment of reporter
molecules to the nonreducing end of cellulose is
nontrivial, and thus, few reliable model substrates exist
for characterization of enzymes exhibiting this specificity.
GH6 family cellulases play a significant role in synergistic
biocatalytic conversion of lignocellulosic biomass, yet despite
decades of careful study, many questions remain as to even the
most basic aspect of their molecular function. The path to
definitively answering these questions of GH6 function will
encounter obstacles including those common to fungal GHs
such as high-throughput heterologous expression and character-
ization as well as the unique aspects related to the GH6 catalytic
mechanism. We anticipate the development of reporter
molecules for nonreducing end specific CBHs on either
cellulose or soluble substrates will represent one of the most
meaningful advances toward understanding GH6 catalytic
function. A recent publication demonstrated a step toward
this goal while also acknowledging the possibility that more
advanced reporter molecule substrates may exist.704 These types
of substrates not only are capable of elucidating the molecular
underpinnings of GH6 function; model substrates will also
prove useful in understanding the extent of processivity and for
selection of enzymes for maximum synergistic function as part of
biomass conversion cocktails. Furthermore, development of
GH6 variants in the search for thermal stability and/or activity
improvements will profoundly benefit from a more accurate
assessment of enzyme performance.
8. FAMILY 5 GLYCOSIDE HYDROLASES
GH5s have emerged as key EGs in biomass conversion cocktails.
GH5 members were originally referred to as family A cellulases
when some 21 β-glycanases were categorized into six different
families using hydrophobic cluster analysis to group similar
sequences.439 The two original fungal members of family A were
EG III from T. reesei (TrCel5A) and EG I from Schizophyllum
commune. Other members of this family were under
investigation at that time,728,729 though without a known
sequence, inclusion of these enzymes in the family A
classification would not come until many years later. As a
growing volume of sequence data became available, the
nomenclature expanded to include hydrolytic activity beyond
that of β-glycan hydrolysis, and family A cellulases became
known as family 5 GHs.654 Today, the GH5 family is one of the
largest and most diverse families of GHs with over 4000
entries.151,153
Though a single family designation, GH5s exhibit quite a large
degree of variation in specificity and hydrolytic activity likely as a
result of divergence from a common ancestor.730,731 In addition
to 1,4-glucanase activity, the GH5 family includes enzymes with
reported 1,6-galactanase, 1,3-mannanase, 1,4-xylanase, and
xyloglucanase activities. Along with 18 other GH families,
GH5s belong to the GH-A clan exhibiting a (β/α)8 fold
1383
Review
characteristic of enzymes capable of degrading equatorially
oriented glycosidic linkages.730,732,733 More specifically, the GH-
A clan, and thus GH5s, belong to the 4/7-superfamily of βα-
barrel glycosidases.730,731 Within this superfamily, the retaining
mechanism is catalyzed by the conserved glutamate at the ends
of β-strands 4 and 7, though very little sequence homology is
otherwise observed.730 Given such a broad categorization of
low-homology enzymes based on the (β/α)8 fold, significant
variety emerges in enzymatic behavior with at least 20 different
experimentally determined enzyme classes within this family
alone.734 A recent study revisiting GH5 classified structures
given newly available structural data even went so far as to
recategorize several enzymes as GH30s, illustrating the difficulty
of sufficiently describing enzymatic activity based on sequence
data or fold alone.735
Within the GH5 family, there are also 51 currently recognized
subfamilies that have been defined by phylogenetic analysis with
several undefined subfamilies anticipated as sequence data
continues to grow.734 Subfamily classification in GH5, or
cellulase family A, began in 1990 with five original subfamily
classifications proposed, A1−A5.736 Five additional subfamilies
were defined in the 15 years that followed until it became clear
that a robust sequence-based subfamily classification approach
was possible.734,737−740 Still, these newly defined phylogenetic
subfamilies encompass both variety of specificities and species
within the subfamily classification. Of the 51 identified GH5
subfamilies to date, experimentally categorized cellulolytic
behavior (EC 3.2.1.4 and EC 3.2.1.91) has been observed in
subfamilies 1, 2, 4, 5, 22, 26, 37, and 39 representing bacterial,
archaeal, and eukaryotic taxonomies. Given the vast body of
GH5 data available as well as the scope of this review, we focus
our discussion of GH5 literature on fungal GH5s exhibiting
cellulolytic behavior except where discussion of bacterial GH5
mechanisms provides relevant mechanistic insights.
Saloheimo et al. reported the first isolation and sequencing of
the egl3 gene coding for T. reesei EGIII.323 The total mass of
EGIII was estimated at 49.8 kDa and was described as having
modular organization as with other T. reesei cellulases
characterized at that point. The 35 amino acid N-terminal
region exhibited particularly high sequence homology with that
of CBHII and the C-terminal regions of CBHI and EGI, which
we now know derives from the characteristic T. reesei cellulase
modular appendage of a family 1 CBM (see section 5). At nearly
the same time, Ståhlberg et al. reported the isolation of the
EGIII core domain from the 61-residue N-terminus region. The
authors described the N-terminal domain as a heavily
glycosylated structural element common to the other T. reesei
cellulases and posited that modular organization may be a
characteristic of all Trichoderma cellulases.741 Saloheimo et al.
also identified EGIII as a glycoprotein, predicting one N-
glycosylation site on the basis a surface exposed Asn-Phe-Thr
motif and a great deal of O-glycosylation.323 EGIII was shown to
cleave reducing sugars from cellodextrins, though at a rate 50−
200 times slower than that of T. reesei EGI. Comparison of the
new EGIII sequence with that of an unpublished S. commune
EGI sequence indicated 30.4% sequence identity,323 and while
this seems low at first glance, it would later become apparent
that this family of cellulases is only loosely linked by sequence
homology. Saloheimo et al. also noted that a protein previously
identified by Shoemaker and Brown in 1978, EG IV,728,729 was
likely actually EGIII, as the amino acid compositions were
remarkably similar.
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The T. reesei family 5 EG is responsible for a significant
portion of the fungi’s EG action, with some reports estimating
the EG contribution at nearly 55% of total EG activity.742 As
such, this EG is often incorporated as a key component in many
industrial biomass conversion cocktails. Over the past four
decades, a great deal of effort has gone into characterizing its
action and defining the limits of stability by many different
groups. Importantly, we note that over this time this particular
enzyme has been referred to in the literature by at least three
different names. The EGIII moniker was given upon the original
characterization by Saloheimo et al.323 Earlier, a report of a
partial amino acid sequence determined by Edman degradation
assigned the enzyme EGII.632 It eventually became clear that
these were the same enzyme. Up until the late 1990s, most
publications referred to this enzyme as EGIII, but more recently,
the enzyme has frequently been referred to as EGII. As GH
nomenclature evolved, the enzyme has also been referred to as
Cel5A. To avoid confusion throughout the remainder of this
discussion, we adopt the modern convention of Cel5A with
reference to the T. reesei family 5 EG. A summary of GH5
structures that are discussed in this section is provided in Table
10.
Table 10. Reported Fungal GH5 Crystal Structures
source and original
name in primary
citation PDB code resolution (Å) brief highlights
Thermoascus 1GZJ 1.62 first fungal GH5
aurantiacus Cel5A structure
reported
1H1N 1.12 apo wild-type
Piromyces rhizinf lata 3AYS 2.20 complex with
Egl1 cellotetraose
Trichoderma reesei 3QR3 2.05 apo wild-type
Cel5A
8.1. Structural Studies
The first solved structure of a GH5 enzyme was of the bacterial
C. thermocellum EG CelC in 1995.743 Dominguez et al. observed
that the general fold corresponded to the (β/α)8 barrel topology
first observed in triose phosphate isomerase and is referred to as
a TIM barrel. This (β/α)8 tertiary structure is by far the most
common observed fold, with an estimated 10% of all known
enzyme structures falling into this class.744,745 It has been
suggested that the variety of (β/α)8 barrel structures emerged as
a result of divergent evolution from a common ancestor, thus
accounting for the vast diversity of sequences and functionality
allowing (β/α)8 barrel proteins to function as hydrolases,
oxidoreductases, transferases, lyases, and isomerases.746
Within the GH5 family, a core set of amino acid residues serve
as distinction from other (β/α)8 barrel hydrolases. These
include the catalytic glutamates, identified previously through
biochemical characterization as well as a handful of other
residues thought to support catalysis or substrate bind-
ing.747−750 Solution of the C. thermocellum CelC structure
captured these residues (Glu280, His198, and Trp313) in an
arrangement suggesting the nucleophilic glutamate is assisted in
catalysis by hydrogen bonding. The cellotriose-bound structure
(3 Å) also illustrated carbohydrate−substrate interactions
confirming Glu140 serves as the nucleophilic donor and that
Asn139 and His90 contact the substrate occupying key positions
near the catalytic glutamates. The vast substrate diversity within
the GH5 family and throughout the over 30 (β/α)8 super-
Review
families likely originates from sequence variations that directly
contribute to both major and minor structural variations.751
These structural variations, such as the two minor β-bulges
located at strands β3 and β7 of C. thermocellum CelC and the 54-
residue subdomain insertion connecting the α6 helix and β6
strands,743 greatly affect overall function and appear to be
related to thermal stability (Figure 53A).752
Many GH5 structures spanning a range of taxonomies and
substrate specificities were solved in the interim between the
appearance of the original CelC structure and the first fungal
GH5 cellulase structure in 2002. Several of these structures from
bacterial cellulases have been invaluable in defining the catalytic
mechanism of GH5 cellulases and will be discussed separately
with respect to catalytic function.206,754,755
8.1.1. Catalytic Function. As members of the GH5 family,
fungal GH5 cellulases hydrolytically cleave the glycosidic
linkages of their substrates using the double-displacement
retaining mechanism, outlined in Section 3.157 Barras et al. first
described this for the bacterial Erwinia chrysanthemi EG Z
identifying the retention of the anomeric carbon product
configuration.756,757 The authors went on to speculate that two
conserved glutamates served as the catalytic acid/base and
enzymatic nucleophile as we will discuss at length below. Much
of the available insight into catalytic mechanisms of GH5s has
been observed in bacterial representatives such as E.
chrysanthemi. Thus, in the following section, we briefly deviate
ref from our focus on fungal cellulases to discuss our current
740 understanding of GH5 catalytic mechanisms in general.
Initial characterization studies identified key catalytic residues
through biochemical methods such as mutagenesis and use of
753
labeled substrates. One of the first studies to investigate the
762
catalytic mechanism of GH5s was conducted in 1990, shortly
30 after some of the first GH5 enzymes were discovered.758 Baird et
al. aligned 16 different amino acid sequences of known
celluloytic enzymes. Though none of the sequences exhibited
greater than 25% similarity to the other sequences in the set, the
multiple sequence alignment identified a recurring three-residue
motif in all the cellulases examined. Baird et al. had uncovered
the Asn-Glu-Pro motif defining in part the catalytic active site of
GH5 cellulases. Site-directed mutagenesis of the Glu to Gln in
two representative bacterial EGs confirmed a detrimental loss of
activity upon mutation. The participation of this conserved Glu
in catalysis was again confirmed in both C. thermocellum and E.
chrysanthemi GH5s.759,760 Without the benefit of structural
insight, these initial studies identified the catalytic acid/base of
GH5 cellulases, though it would not be until later that the Glu
was confirmed as such.
Following shortly after the discovery of the GH5 catalytic
acid/base, the complementary nucleophile, also a glutamate, was
confirmed. Wang et al. used a clever labeling approach to
determine the nucleophilic residue of C. thermocellum CelC
involving radiolabeling the enzyme with tritiated inhibitor and
spectroscopically analyzing cleaved, labeled peptide frag-
ments.747 Macarron et al. had previously speculated this same
glutamate was the catalytic nucleophile in TrCel5A.750 The
family 5 nucleophilic glutamate generally belongs to a Glu-Xxx-
Gly motif, where Xxx is typically an aromatic residue.761
Identification of the conserved acid/base and nucleophile
glutamates served in part as the basis for refining GH
classifications including family A cellulases and ultimately the
development of the family 5 classification.732,733
The double-displacement retaining mechanism is also known
to require a water molecule for nucleophilic attack. Evidence of
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Figure 53. GH5s are among the many proteins exhibiting the relatively common (β/α)8 topology. (A) In 1995, the first GH5 structure, originating
from the bacteria C. thermocellum, was solved.743 The celluloytic EG CenC, in purple cartoon, is shown aligned with the first fungal GH5 cellulase
structure, T. aurantiacus Cel5A in green, solved nearly a decade later.740 (B) The GH5 structures share the same basic topology but exhibit
extraordinary diversity in the loop regions connecting the primary β-sheets and α-helices. A third GH5 cellulase, also from T. aurantiacus in tan, was
solved at nearly the same time as the first fungal Cel5A structure.753 C. thermocellum CenC exhibits a large subdomain insertion between the α6 helix
and β6 strand, where the two T. aurantiacus Cel5A structures exhibit a very compact loop (highlighted by the dashed red circle). The cellotriose ligand
from C. thermocellum CenC is shown in gray stick, and the positioning of the loops relative to the cellulose substrate suggests functionality in substrate
recruiting, though likely using very different mechanisms.

an appropriately positioned water molecule would not come
until later when structural characterization of the B.
agaradhaerens Cel5A reaction pathway was captured by Davies
et al.206 Other residues, including an asparagine, histidine, and
arginine, have been hypothesized to participate in supporting
catalytic roles.740,747 However, it is not clear these interactions
are conserved across GH5s or the precise role each residue
plays.
Structural evidence of the double-displacement mechanism in
GH5s was obtained in 1998 when Davies et al. captured the
entire reaction coordinate in a series of structures from B.
agaradhaerens Cel5A, a β-1,4-glycanase.206 Five different
structures describe the individual states of the reaction
including: the initial apo conformation of the active site, the
Michaelis complex, two versions of the glycosyl-enzyme
intermediate, and the product bound conformation. We
illustrate these states in Figure 54, as this reaction coordinate
is general for fungal GH5 cellulases.
Prior to hydrolysis, the cello-oligomer substrate is initially
bound within the GH5 active site in the catalytically competent
Michaelis complex (Figure 54). The catalytic acid/base, Glu139
for B. agaradhaerens Cel5A, and the nucleophilic glutamate,
Glu228, are positioned over the −1 glucopyranose so as to
initiate catalysis. The proton of the acid/base hydrogen bonds
with the glycosidic oxygen, while the nucleophlic glutamate
hydrogen bonds with the anomeric carbon of the −1
glucopyranose ring. The −1 glucopyranose must adopt an
energetically unfavorable skew conformation (1S3) to correctly
position the leaving group sugar significantly higher than would
otherwise naturally occur. The use of a fluorine-substituted
dinitrophenyl substrate made capture of the intact glycosidic
linkage spanning the −1/+1 binding sites possible.
1385
The first step in the double-displacement hydrolytic
mechanism of GH5s is the glycosylation step in which the
glycosyl-enzyme intermediate is formed, Figure 54. At this
point, a proton from the catalytic acid/base, Glu139, has been
transferred to the leaving group sugar, a non-natural
dinitrophenyl group in the Davies et al. study. The nucleophilic
Glu228, having attacked the anomeric carbon of the −1
glucopyranose moiety, is then covalently bonded to the
oligomeric substrate. The −1 glucopyranose sugar returns to
its energetically preferential 1C4 state. With newfound space in
the enzyme active site upon expulsion of the product sugar and
relaxation of the substrate, a water molecule takes the place of
the glycosidic oxygen. The authors managed to capture two
different versions of the GH5 glycosyl-enzyme intermediate
covalently bonded to a cellobiosyl and a cellotriosyl substrate.
For the most part, the length of the substrate has little effect on
the position of the catalytic residue. However, a conserved
tyrosine residue appears to undergo a conformational change as
a result of the shorter cellobiosyl covalent attachment. This
residue has been identified as a contributor to catalysis,30,743,762
though the exact means by which this occurs remains unknown.
The authors refrain from speculating as to the role of this
tyrosine, though it appears the residue is critical to transition
state stabilization intermittently interacting with the −1 sugar
hydroxyl group and Glu228. Davies et al. do go on to propose
that Arg62 is relevant to the correct positioning of the
nucleophilic glutamate throughout the catalytic cycle.
Finally, the nucleophilic water molecule catalyzes the second
step of the double-displacement mechanism, deglycosylation. A
proton from the water is donated to the catalytic acid/base, and
the remaining hydroxide caps the cleaved glycosidic linkage. The
nucleophilic glutamate is returned to its original negative charge
state, and the enzyme is reset to its original state ready to
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Figure 54. Retaining mechanism of GH5 cellulases illustrated by B. agaradhaerens Cel5A structures captured along the reaction coordinate.206 The
catalytic residues are shown in yellow stick. Glu139 is the catalytic acid/base, and Glu228 is the enzymatic nucleophile. Arg62 and Tyr202 appear to
serve supportive roles. The substrate, representative of β-1,4-linked glucans, is shown in cyan stick. The water molecule that attacks the substrate
anomeric carbon in the deglycosylation step is shown as a red sphere. The Michaelis complex (PDB 4A3H) is shown at left and illustrates the
catalytically competent enzyme−substrate conformation. Two covalently bound glycosyl-enzyme intermediate states following the glycosylation step
of the double-displacement mechanism have been captured (PDB 5A3H and 6A3H), and the length of the product side substrate appears to affect the
conformation of Tyr202. The product, cleaved from the protein following the deglycosylation step, is shown at right (PDB 3A3H).

hydrolyze another glycosidic bond. Generally, most GH5s do
not exhibit any significant degree of processive action. Thus, the
substrate likely dissociates from the active site, and a new
oligomer association forms prior to the next catalytic cycle.
Overall, the Davies et al. study provides a firm structural basis
for understanding the hydrolytic mechanisms of GH5 enzymes,
including cellulases.206 However, it is not immediately clear as to
which of the two steps, glycosylation or deglycosylation, is rate-
limiting in GH5s or if this is consistent across the family.
Currently, our understanding of the rate at which each step
proceeds is limited to theoretical studies of bacterial GH5
cellulases. Liu et al. performed QM/MM simulations of both the
glycosylation and deglycosylation steps of Acidothermus
cellulolyticus Cel5A.763 The glycosyl-enzyme intermediate was
approximated from MD simulations in lieu of a structure-based
approximation. The authors report free energy barriers of 25.7
and 29.4 kcal/mol for the glycosylation and deglycosylation
reactions, respectively. However, the authors caution that the
difference between the two barriers may not be statistically
significant given the relatively low level of theory applied.
Furthermore, the reported barriers are likely unreasonably high
as a whole, again likely as a result of the level of theory. Similarly,
Saharay et al. apply QM/MM to examine deglycosylation in B.
agaradhaerens Cel5A obtaining a free energy barrier of 24.2
kcal/mol.764 Saharay et al. do not provide explanation as to the
differences in their calculations and those of Liu et al. despite
having used the same level of theory. In general, our
1386
fundamental understanding of catalytic mechanisms in this
family lags behind that of GH7 and offers an area for continued
investigation.
In the sections that follow, we focus on the four fungal GH5
cellulase structures solved to date noting that difficulties with
protein expression and purification have hindered accumulation
of a large fungal structural data set (Table 10).765 A multiple
sequence alignment of the four fungal structures alongside the
bacterial B. agaradhaerens Cel5A sequence is provided in Figure
55 to aid in comparative discussion.
8.1.2. T. aurantiacus Cel5A. Lo Leggio and Larsen
reported the first fungal GH5 cellulase structure, a 1.62 Å EG
from T. aurantiacus (PDB 1GZJ, Figure 53A).740 In addition to
being the first fungal cellulase structure, the TaCel5A structure
documented the first structural representative of the GH5
subfamily 5 classification (formerly A5/6). As with previous
GH5 structures, the canonical (β/α)8 fold comprises the general
tertiary structure. However, the TaCel5A structure is remark-
ably compact with few extensions to the loops connecting the α
and β regions (Figure 53B).
The TaCel5A structure does not include a bound ligand, but
on the basis of appearance, Lo Leggio and Larsen suggest the
EG may be capable of binding up to a celloheptaose oligomer
within the −4 to +3 binding sites. The enzyme active site is
formed by a wide and shallow groove lined with aromatic
residues Trp278, Trp279, Trp273, Trp170, Trp174, and Tyr200
(Figure 56A). This aromatic-mediated substrate recognition
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Figure 55. Sequence alignment of the four fungal GH5 structures discussed here alongside the bacterial B. agaradhaerens Cel5A (BaCel5A) from
which catalytic function was elucidated. Notably, the sequence alignment illustrates the relatively low sequence similarity among the members of this
GH family. Strictly conserved residues are shown in red block, and chemically similar residues in red text. The blue boxes indicate chemical similarity
across a grouping of residues. The secondary structural element of TrCel5A and BaCel5A are shown above and below the sequences, respectively. The
catalytic acid/base motif is shown in a pink box, and the catalytic nucleophile is shown in a yellow box. The figure was generated with ESPript (http://
espript.ibcp.fr).347

mechanism is typical of GHs,766 yet of the tryptophan residues
lining the active site, only Trp273 in the −1 binding site is
strictly conserved. However, aromatic residues near the +1 and
+2 binding sites are spatially conserved throughout GH5s
suggesting functional relevance. The spatially conserved
tryptophan in the +1 binding site of TaCel5A is Trp170.
1387
Chemical modification of the homologous residue in TrCel5A
decreased kcat/Km by nearly half.749 The authors hypothesize this
residue serves a configurational function positioning the
glycopyranose moiety in the −1 binding site in the nonenergeti-
cally favorable skew conformation necessary for catalysis. The
tyrosine residue, Tyr200, located in the −2 binding site of
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Figure 56. (A) First fungal GH5 cellulase structures, both T. aurantiacus Cel5A, exhibit an extensive network of aromatic residues comprising the wide
and shallow active site groove.740,753 The aromatic residues, shown labeled in yellow stick, serve to mediate carbohydrate substrate interactions.
Surprisingly, only Trp273 in the −1 binding site and the catalytically functional Tyr200 are strictly conserved among the aromatic residues. However,
many others are spatially conserved by chemically similar residues. (B) A handful of conserved residues, cyan stick, are associated with GH5 cellulolytic
function and differentiate these (β/α)8 structures from the thousands of others. Several, including Tyr200, Glu133, and Glu240, directly participate in
catalysis. Other conserved residues contribute to structural stability through disulfide bonds, orange stick, and salt bridges, magenta stick.

TaCel5A, is also strictly conserved. Prior evidence suggests that
the tyrosine hydroxyl group may participate in catalysis through
interaction with the nucleophile rather than through direct
stacking interaction with the carbohydrate moieties.206,743,747
Outside the aromatic residues, Lo Leggio and Larsen put
forth functional roles for several of the conserved residues.
Through structural comparison, the authors identified con-
served residues including two glycines (Gly8, Gly44), the
catalytic glutamates (Glu133, Glu240), as well as the six
canonically conserved active site residues (Asp132, Arg49,
His93, His198, Tyr200, Trp273). Conservation in GH5s is
almost entirely restricted to the active site, as shown in Figure
56B. The exceptions to this in TaCel5A are the two glycine
residues and Arg49, whose functions remain unclear. The
authors suggest Gly44 is a part of the Schellman C-terminal
capping motif conserved throughout GH5s.767 The functional
relevance of Gly8 is less obvious, as this residue lies in the
middle of the β1 strand away from the substrate and
conservation is unnecessary to maintain the (β/α)8 fold.
Arg49 is located in the β2 strand. This arginine is conserved in
all members of the 4/7 superfamily except those of GH10 and
GH26. As with Gly44, this conserved residue is seemingly
related to the Schellman C-terminal capping motif, as the motif
is also not conserved in GH10 and GH26.
The positions of the catalytic residues in the TaCel5A
structure were also informative. As is often the case, the
crystallization conditions under which the protein was crystal-
lized, pH 9.0, were far from the optimal acidic pH of
approximately pH 4.0. Additionally, the structure did not
contain a ligand bound in the active site. Despite these
limitations, the catalytic residues were found in a catalytically
competent position,740 making superimposition of cello-
oligomers from other bacterial GH5 structures straightforward
and informative from a mechanistic perspective. The positioning
of these residues under adverse conditions may be related to the
apparent general flexibility of the GH5 EG active site as was
1388
observed for B. agaradhaerens Cel5A.206 More broadly,
mounting structural evidence such as this and recent GH18
studies suggests flexibility of the catalytic residues is a hallmark
of endo-active enzymes in general.206,768
A second even higher resolution (1.12 Å) TaCel5A structure
followed almost immediately after the first (PBD 1H1N), again
without a bound substrate.753 Van Petegem et al. offer additional
molecular level insights not explicitly discussed by Lo Leggio
and Larsen.740 The aromatic lined active site groove of TaCel5A
was again noted, but Van Petegem et al. describe an additional
aromatic residue (Phe16) that may participate in carbohydrate
stacking interactions in the −2 binding site. Though not
explicitly described as such, perhaps the most interesting
findings from the TaCel5A structure reported by Van Petegem
et al. is the discussion of structural details that likely contribute
to the thermal stability of this EG. Lo Leggio and Larsen report a
single disulfide bond between Cys212 and Cys249 noting that it
does not appear to be conserved among GH5s. Contradictorily,
Van Petegem et al. describe the apparent conservation of the
same disulfide bond in 20 different GH5 EGs. In both studies, it
is unclear which subset of enzymes was used in the sequence
comparisons making it difficult to speculate why conservation of
the disulfide bridge appears inconsistent. Nevertheless, disulfide
bonds have long been associated with thermal stability and may
contribute to the same in TaCel5A.769−771 A salt bridge between
Arg154 and Asp188 is also reported, though not as strictly
conserved. The Arg154 residue is located toward the end of the
α4 helix, and Asp188 is located in a largely unstructured, solvent-
exposed loop connecting the α5 helix to the β6 strand. It is
tempting to surmise that the presence of the salt bridge in a
region otherwise susceptible to denaturation contributes some
measure of stability, as, like disulfide bridges, the appearance of
salt bridges in thermophilic proteins has been implicated in
stability.772−775
Van Petegem et al. also very briefly discuss the function of the
loop connecting the α6 and β6 structural elements. Specifically,
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the authors describe a potential substrate positioning mecha-
nism based on both an extensive observed hydrogen bonding
network in this region as well as superimposition of the T.
aurantiacus structure with the cellobiose-bound C. thermocellum
CenC (Figure 53B). While proximity of this loop to the C.
thermocellum CenC cellobiose does suggest function, little else
can be said regarding the mechanism given the significant
structural deviations between the two EGs in this region. C.
thermocellum CenC exhibits an impressive subdomain insertion
represented by a very compact, unstructured loop in TaCel5A. It
is difficult to imagine these two drastically different features
behave in a similar substrate-recruiting fashion.
8.1.3. P. rhizinf lata EglA/CelA. The first fungal GH5
cellulase structure exhibiting a bound ligand was solved by
Tseng et al. in 2011.762 The E. coli expressed EG from P.
rhizinflata, described in literature as both EglA and CelA
(PrEglA), captured the orientation of a cellotriose molecule in
the −3 to −1 binding subsites at 2.2 Å resolution (PDB 3AYS).
The authors reported that the N-terminal His tag attached for
purification prevented soaking in a cellulose ligand by blocking
the active site. Cocrystallization with the catalytically inactive
PrEglA (E154A) was successful, however. These limitations
likely apply to other fungal GH cellulases explaining in part the
long delay in solution of a substrate-bound structure. The
authors also solved the complementary apo wild-type structure
at 2.0 Å resolution (PDB 3AYR) illustrating that the enzyme
underwent little to no change as a result of substrate binding.762
PrEglA is similar to the TaCel5A structure in several ways
including the presence of several spatially conserved aromatic
residues lining the active site and the presence of a single
disulfide bond. Interestingly, the disulfide bond in PrEglA
(Cys27 to Cys43) is located in a different loop from that
observed in TaCel5A connecting the β1 strand to the α1 helix on
nearly the opposite side of the protein near the −3
glucopyranose of the bound cellotriose (Figure 57). The PrEglA
disulfide bond almost certainly plays an indirect role in substrate
Figure 57. Active site of P. rhizinf lata EglA, the first fungal GH5
cellulase structure to capture the position of a bound ligand. The
cellotriose-bound variant structure (3AYS) is shown in aquamarine
cartoon and stick, and the apo wild-type structure (3AYR) in light pink
cartoon and stick. Aromatic residues, Trp44 and Tyr231, are shown in
yellow stick, and the cellotriose is shown in gray stick. Water molecules
are shown as red spheres.
1389
Review
positioning given its proximity to Trp44, which is positioned
directly over the −3 glucopyranose moiety. This suggests that
while both PrEglA and TaCel5A use the same catalytic
mechanism to hydrolyze the glycosidic linkage, the two enzymes
make use of a different molecular level mechanism for substrate
recruitment.
The PrEglA structure also explicitly illustrated molecular
phenomena related to catalysis in fungal GH5 cellulases. Tseng
et al. note six water-mediated hydrogen bonds between the −1
and −2 glucopyranose moieties in the 3AYS structure (Figure
57).762 The water near the anomeric carbon of the −1
glucopyranose hydrogen bonding with Glu154 has been
implicated as a participant in the catalytic mechanism of
GH5s, specifically as a means of nucleophilic attack on the
glycosyl-enzyme intermediate.206 The PrEglA ligand structure
captures the water molecule near the −1 glucopyranose despite
being catalytically inactivated by an E154A mutation. Super-
imposition of the Glu154 side chain from the wild-type structure
with E154A confirms the catalytic acid/base points toward the
O1 atom of the −1 glucopyranose at 1.7 Å, and the
complementary nucleophile, Glu278, hydrogen bonds with the
anomeric carbon of the −1 glucopyranose at 3.2 Å. This catalytic
arrangement of residues, including nucleophilic attack by the
water, is in line with that previously observed in a bacterial GH5
cellulase. 206 The conserved Tyr231 was also observed
participating in an apparent catalytic arrangement by hydrogen
bonding with the Glu278 side chain.
The PrEglA cellotetraose-bound structure offers an intriguing
explanation for the enzyme’s putative processive ability. The
authors purport, on the basis of previous biochemical assays,
that PrEglA is “both an endoglucanase and a cellobiohydrolase”,
which we take to mean that the enzyme exhibits both processive
and nonprocessive behavior.776 The authors do not attempt to
further connect structure to function in an enzyme that certainly
resembles an EG by all appearances. However, Tseng et al.
indicate the bound cellotetraose ligand is nearly completely
surrounded by protein at the −1 binding subsite with the
remainder of the active site exposed to water. This observation is
likely directly related to the observed processive ability of the
enzyme in both increasing ligand binding free energy and
maintaining contact with crystalline substrates allowing
processive hydrolytic events.580
Finally, the authors observe what they consider to be a
potentially dimeric structure in which the O1 atom of the −1
sugar appears to hydrogen bond (3.2 Å) with the OE2 of the
symmetry-related Glu242.762 Site-directed mutagenesis of
E242A indicated overall catalytic function was retained,
suggesting dimeric catalysis by the proposed mechanism was
unlikely. Nevertheless, the authors go on to suggest that because
the first 100 amino acids encoded by the PrEglA cDNA
fragment were similar to the last third of the (β/α)8 barrel
sequence that PrEglA comprises at least two CDs acting in
tandem. However, the structural evidence to date does not
support this hypothesis.
8.1.4. T. reesei Cel5A (Formerly EG II/EG III). The most
recent fungal GH5 cellulase structure originates from T. reesei
(PDB 3QR3).30,566 The enzyme, Cel5A or formerly EGII/
EGIII, accounts for up to 55% of the total EG activity of T. reesei
and thus is a key component of many industrial biomass
conversion cocktails.742 Thermal stability of the enzymes within
these cocktails is key to effective deployment in commercial
processes and has been a primary focus of recent protein
engineering efforts.324,752,777−779 The thermal stability of
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Figure 58. First structure of TrCel5A reveals key details regarding disulfide bonds and loop insertions that may aid in understanding contributions to
thermal stability in GH5 EGs. (A) The TrCel5A structure, slate cartoon, is shown aligned with the homologous TaCel5A structure, green cartoon.
Primary differences in the two structures appear in the noted loop regions connecting the primary structural elements. TrCel5A also exhibits a β-
hairpin element, red cartoon, with a tryptophan residue that stacks against the protein face. (B) TrCel5A exhibits four disulfide pairs, orange stick, that
connect primary structural elements, yet surprisingly may not significantly contribute to thermal stability. (C) The active site of TrCel5A contains
several conserved residues, gray stick, that may contribute to stability of transition states in catalysis or substrate binding. The catalytic acid/base,
Glu218, and the nucleophile, Glu329, are shown in yellow stick.

TrCel5A (reported Tm of 69.5 °C) is relatively low compared to
other hyperthermally stable GH5s and is a likely target for
stability improvement. Lee et al. solved the 2.05 Å apo structure
of TrCel5A to uncover the molecular level contributions to
thermal stability by comparison; however, the structure
uncovers as many questions as it answers.566
The TrCel5A structure’s closest homologue is TaCel5A with
29% sequence identity and 69% sequence similarity. Compar-
ison of TrCel5A with the TaCel5A structure,740,753 a hyper-
thermally stable enzyme with a Tm of nearly 81 °C,780 can
feasibly provide insight into the variation in molecular features
contributing to reduced thermal stability in TrCel5A. At first
glance, primary differences in the two structures include
extended loops connecting the β1 sheet to the α1 helix, the β3
sheet to the α3 helix, and the α5 helix to the β6 sheet (Figure
58A). The TrCel5A structure also features a protruding β-
hairpin element at residues 378−385 near the final α8 helix,
wherein Trp384 stacks the β-hairpin against the globular face of
the protein (Figure 58A). The functional role of the β-hairpin
remains unknown. However, β-hairpins have been observed in
other bacterial GH5s including Thermotoga maritima Cel5A781
and Clostridium cellulovorans EG D (PDB 3NDY, unpublished).
Though Lee et al. do not investigate the contributions of the
loop insertions to reduced thermal stability, it is tempting to
hypothesize these increasingly disordered regions contribute in
part to a reduced Tm relative to TaCel5A. Recent computational
investigations of a related enzyme lend credence to this
hypothesis, though it remains largely untested experimen-
tally.752
Disulfide bonding in TrCel5A is also a key differentiator from
the homologous TaCel5A. TaCel5A exhibits a single disulfide
bond pinning the α6 to β6 loop to the top of the α7 helix.740,753
PrEglA also exhibits a single disulfide bond and is a known
hyperthermophilic enzyme.762 In contrast, TrCel5A contains 8
cysteine residues forming four disulfide bonds in the protein
(Figure 58B). One of disulfide bonds, between Cys302 and
Cys338, corresponds to that observed in TaCel5A. The disulfide
bond between Cys86 and Cys92 tethers the C- and N-terminal
regions of the β1 to α1 loop. Oddly, the other two disulfide
1390
bonds, Cys162 to Cys169 and Cys343 to Cys393, anchor (β/
α)8 barrel structural elements directly including the α2 helix to
β3 sheet and the α7 helix to the α8 helix, respectively. Intuitively,
one would expect that, with the increased number of disulfide
bonds, TrCel5A would exhibit a greater degree of structural
stability and thus increased thermal tolerance over TaCe-
l5A.769−771 However, on the basis of the structural evidence
presented by Lee et al.,30 there does not appear to be a direct
correlation of disulfide bonds and thermal stability. The authors
report site-directed mutagenesis of several cysteines to serines
resulted in insoluble protein expression in the E. coli
recombinant host. Thus, there is little evidence by which to
ascertain the contributions to thermal stability imparted by each
nonconserved cysteine pair.
Finally, Lee et al. observe catalytic residue orientations in line
with the proposed GH5 catalytic mechanism.30,206 The catalytic
residues Glu218 and Glu329 are separated by a distance of
approximately 5 Å measured from the terminal oxygen atoms
consistent with reports of catalytic residue flexibility (Figure
58C).206 The authors suggest residues Thr328, His288, and
Glu218 function simultaneously to raise the donor carboxylate
pKa promoting efficient catalysis. The nucleophilic glutamate,
Glu329, is observed hydrogen bonding to the OE2 atom of
Arg130 and the OE1 atom of Tyr290 as part of the retaining
catalytic mechanism. As with T. aurantiacus,753 conserved
residues His174 and Trp362 are reported to participate in
substrate binding rather than directly in the catalytic mechanism
despite their proximity to the active site.
8.2. Characterization of Activity and Specificity
In general, GH5 celluloytic function can be classified as endo-
initiated, with only 2 of the 4395 members classified as having β-
1,4-cellobiosidase activity (EC 3.2.1.91), one from C.
thermocellum and one from Teredinibacter turnerae. In the
following sections, we briefly describe the wealth of biochemical
characterization data available for fungal GH5 cellulases. Each of
the enzymes described below have been classified as endo-β-1,4-
glucanases (EC 3.2.1.4) with some purported to exhibit
moderate degrees of processive action.
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8.2.1. T. viride EG III. Some of the earliest reported
biochemical characterizations of GH5s came from T. viride
several years before cellulase classification would relate their
function to other GH5s. In 1978, Shoemaker and Brown
submitted two simultaneous reports on the discovery and
characterization of a set of four EGs from T. viride
demonstrating purity with SDS-PAGE.728,729 Up to this point,
studies had purified fungal EGs (not necessarily GH5s) from T.
viride, T. koningii, Fusarium solani, Sporotrichum pulverulentum,
and Irpex lacteus, though homogeneity of these early
purifications was uncertain.782−785 Shoemaker and Brown
described what was ultimately the first biochemical character-
ization of pure fungal GH5 cellulases, T. viride EGIII and EGIV,
both from subfamily 5. The enzymes were reported as 52 and
49.5 kDa, respectively.729 Specific activity assays suggested the
EGs were active on CMC, PASC, and cellotriose and higher
oligosaccharides with limited Avicel activity.728 Both EGs were
reported to have pH optimums of 4.0 to 4.5 on PASC and CMC
and a noted intolerance for high pH.729
The T. viride cellulases, both EGs and processive cellulases
from other GH families, formed the basis of a commercial
enzyme preparation, Maxazyme CL, from the Dutch company
Gist-brocades (now owned by DSM) and another from Miles
Laboratories. After the report from Shoemaker and Brown,
several years passed before any additional characterization
occurred. In 1985, Beldman et al. reported the purification and
characterization of the commercial Maxazyme preparation,
though much of the report focuses on the processive cellulase
function.786 The authors followed up their characterization
efforts with a description of the apparent synergy of the T. viride
cellulases, which relies significantly on the presence of EGs such
as EGIII and EGIV.787 After these initial reports, much of the
literature related to T. viride GH5 EGs surrounds strain
engineering for enhanced production with a few reports of
recombinant expression for improved stability and pH
optimum.
8.2.2. TrCel5A. T. reesei (now also known as Hypocrea
jecorina) Cel5A (TrCel5A) is a model fungal GH5 EG
(subfamily 5) having found application in some modern
cellulase-based commercial enzyme preparations.788 As such,
much of the fungal GH5 characterization efforts to date have
focused on this enzyme. Early characterization focused on
delineating activity between the suite of T. reesei cellulases and
broadly describing specificity. In 1985, van Tilbeurgh and
Claeyssens used 4-methylumbelliferyl-β-D-glycoside substrates
in an attempt to explicitly differentiate T. reesei CBHs, EGs, and
β-glucosidase activities.301 Notably, TrCel5A was the only
cellulase to cleave the fluorescent phenol group from the
fluorogenic 4-methylumbelliferyl-β-D-cellotrioside. This techni-
que would later be used to characterize relative activities of
commercial enzyme preparations, the results of which high-
lighted the need to pay close attention to the model substrates
used in evaluation of overall activity and synergism, as it is now
clear that it is impossible to differentiate CBH from EG activity
using artificial fluorogenic substrates such as 4-methylumbelli-
feryl-β-D-glycoside.789
In 1987, Penttilä et al., having expressed and cloned the egl3
gene encoding TrCel5A, recombinantly expressed the EG in S.
cerevisiae in an effort to understand the effects of expression host
on activity.283 The authors found that while the recombinant
enzymes remain active, the expression host affects both
specificity and enzyme morphology. The change of morphology
resulting from heterologous expression of TrCel5A compared to
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the control strain was speculated to result from hyper-
glycosylation common in yeast expression.
As previously mentioned, Saloheimo et al. described what
appears to be the first isolated and sequenced fungal GH5
cellulase.323 Alongside this isolation, the authors characterized
the activity of the new EG, as its sequence differed substantially
from the known cellulases at the time, TrCel6A, TrCel7A, and
TrCel7B, indicating potentially new function. TrCel5A, like
TrCel6A, did not hydrolyze cellobiosides or lactosides, and thus,
the authors speculated specificity was similar. Saloheimo et al.
also reported a pH optimum of 4.0−5.0 on a cellotrioside
substrate at 50 °C, comparable to the T. viride EGs.
A flurry of activity surrounding the mode of TrCel5A action
occurred in the early 1990s with a series of papers uncovering
the catalytic nucleophile,750 described above, putative binding
models,748 essential aromatic residues,749 and a high degree of
endo-activity.304 With a basic understanding of TrCel6A and
TrCel7A specificity and action in place at this point, Macarrón et
al. set out to develop a similar level of understanding of TrCel5A
action. Having purified the TrCel5A with an attached CBM, the
authors report TrCel5A exhibits a stable pH range 4.0−6.3 at 55
°C, retaining 90% of its activity at 65 °C for 30 min.748
Chromophoric substrates were effectively used to identify
TrCel5A as having a double-displacement catalytic mechanism,
the first report of such. In the case of cleaving the 2-chloro-4-
nitrophenol bond, the deglycosylation step was reported as the
rate-limiting step, though nucleophilic competition experiments
between methanol and water point to the glycosylation step as
rate-limiting. Ultimately, no conclusion can be made regarding
rate-limitation in the double-displacement mechanisms from the
reported data. However, Macarrón et al. were able to propose a
five-subsite binding model for TrCel5A based on observed
cleavage products (Figure 59). To date, this remains to be
validated from a structural standpoint, with only a single apo
structure of the enzyme reported.30
Macarrón et al. later investigated the function of three
tryptophan residues in binding and catalysis using N-
bromosuccinimide modification.749 Given the likely role of
conserved aromatic residues in carbohydrate substrate binding,
the authors hypothesized chemically modified tryptophans
Figure 59. Proposed five-subsite binding model for TrCel5A, illustrated
with two possible hydrolysis pathways for 4-methylumbelliferyl-β-D-
cellotrioside, with the open rectangles indicating D-glucosyl moieties
and filled rectangles indicating 4-methylumbelliferyl moieties. The
lettered, indented block represents the binding site of TrCel5A.
Catalysis takes place between the C and D subsites, as indicated by the
arrows indicating catalytic protein residues. The possibility of methanol
transfer as part of the EG activity is indicated. Reprinted with
permission from ref 748. Copyright 1993 the Biochemical Society.
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would affect binding and hydrolysis. Chemical modification was
able to alter three tryptophans in total: one putatively part of the
CBM, one likely very near the catalytic active site, and one
unspecified modification not affecting activity or binding. The
modification of the CBM-located tryptophan, mostly likely
Trp5, reduced absorption on Avicel by nearly 50% (see section
5). As TrCel5A is an EG, it is unclear whether this modification
would significantly affect enzymatic activity in practice. The
“active site” tryptophan, thought to be Trp255, reduced rates of
hydrolysis on small soluble substrates, though primarily through
its influence on substrate binding rather than directly through
catalysis. This finding is directly related to the primarily endo-
active function of TrCel5A.304
In later years, the importance of choice of model substrate and
kinetic screening tools became an important emphasis in
cellulase research. Frequently, TrCel5A was included in these
studies, while TrCel7A or TrCel6A was the primary focus. In
developing the now ubiquitous disodium 2,2′-bicinchoninate
assay to quickly assess initial reducing end sugar production
from cellulases, Johnston et al. evaluated Michaelis−Menten
kinetics of TrCel5A relative to the three other primary
Trichoderma cellulases providing a consistent comparison of
kinetic data accounting for initial rates of the biphasic
systems.790 Kipper et al. similarly worked toward an accurate
characterization cellulase kinetic action and, in doing so,
reported the remarkable differences in TrCel5A endo-activity
versus TrCel6A and TrCel7A. Related processive character-
ization efforts also served in verifying the nonprocessive nature
of TrCel5A.315 Jäger et al. investigated the merits of using an α-
cellulosic substrate in the selection of cellulases for alkaline-
pretreated biomass and briefly discussed TrCel5A action on this
realistic substrate.791
Knowing that TrCel5A is a multimodular enzyme consisting
of a CBM attached via a glycosylated linker to the catalytic
domain, a natural follow-up question is to what extent does the
CBM play a role in the enzyme’s action. Nidetzky et al., noting
the inherent difficulties associated with appropriate selection of
binding site models, set out to develop realistic relationships
between cellulase binding and rate of substrate hydrolysis.792 As
part of this study, the authors also evaluated the effects of the
CBM on binding and hydrolysis. After incubation with filter
paper substrate and fluorometric assessment of activity,
Nidetzky et al. developed Langmuir isotherms describing filter
paper adsorption of all the major Trichoderma cellulases.
Relative to TrCel5A, the authors found that (1) the extent of
adsorption increases with agitation, facilitating mass transfer to
the solid substrate, (2) nearly 4 times as many binding sites were
available for the intact TrCel5A compared to the CD alone, and
(3) the relationship of binding to hydrolysis was nonlinear,
suggesting nonproductive binding. These results indicate that
the CD alone does little of the work in maintaining enzyme/
substrate association, and thus, the CBM is critical to effective
hydrolysis at relatively low substrate concentrations.
The effect of CBM substrate binding to real biomass has also
been examined to much the same result as described by
Nidetzky et al. for filter paper.792 Palonen et al. compared the
binding behavior of TrCel7A and TrCel5A both with and
without CBMs on steam pretreated softwood and lignin.793
Again, separation of the catalytic core domains from the CBMs
significantly decreased binding of the enzymes to the substrates,
though the effect on hydrolysis as a result was not determined.
An interesting finding from this work was that the TrCel5A CD
appears to adsorb to the alkaline isolated lignin to a much
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greater extent than TrCel7A. This is likely related to the
characteristics of the globular surface of TrCel5A, as it has been
repeatedly reported that addition of bovine serum albumin
(BSA) in assays enhances stability of TrCel5A.323,792,793 Here,
addition of BSA significantly reduced the binding of TrCel5A to
lignin.
In a related study, Le Costaouëc et al. examined the effects of
CBMs on hydrolysis for TrCel7A and TrCel5A as well as
TaCel7A and TaCel5A on pretreated wheat straw and
spruce.794 TaCel7A and TaCel5A, unlike the Trichoderma
cellulases, naturally lack the CBM module and presumably have
evolved in such a way as to maintain similar levels of hydrolysis.
The authors genetically modified the Thermoascus cellulases
with attached CBMs for a direct comparsion to intact TrCel7A
and TrCel5A. A general conclusion from this study was that, at
high substrate loadings, all enzymes performed better without
the attached CBMs, mostly likely by virtue of the high
population of free oligosaccharides reducing the need for
substrate recognition by the CBM. The Thermoascus cellulases
have likely evolved to function in high substrate loading
contexts, as both enzymes outperformed the CBM deficient
Trichoderma cellulases. Le Costaouëc et al. also suggest that the
CBM−lignin interactions may have detrimental nonproductive
binding interaction that would be mitigated by the removal of
the CBM entirely. Thus, even for Trichoderma cellulases,
removal of the CBM for high substrate loading applications may
result in better overall performance.
Early characterization efforts were particularly successful in
predicting extent of glycosylation in TrCel5A. Both Saloheimo
et al. and Ståhlberg et al. described TrCel5A putatively
exhibiting one N-linked glycan and heavy O-linked glycosylation
on the N-terminal structural domain, i.e., the CBM and linker
(as described in section 5).323,741 Nearly 15 years later, Hui et al.
would follow up these hypotheses to firmly establish the nature
and location of N-linked glycans and the extent of heterogeneity
in O-linked glycans of the linker domain.418 Using capillary
liquid chromatography-electrospray mass spectrometry and
matrix-assisted laser desorption and ionization time-of-flight,
Hui et al. characterized the glycosylation patterns of all four
major Trichoderma cellulases purified from a T. reeei RUT-C30
strain fermentation broth. The authors confirmed that TrCel5A
exhibits a single N-linked GlcNAc at Asn103, the previously
predicted location. 323,418 The glycan is likely trimmed
endogenously after secretion from a higher mannose form.
The authors also indicate the linker exhibits anywhere from 32
to 42 O-linked mannose residues, only slightly fewer than the
39−46 attached to the long, glycosylated TrCel6A linker. The
O-linked glycan moieties have been shown to play a key role in
enzyme substrate binding, and such a high degree of
glycosylation as reported here may account in part for the
significant differences in adsorption between the intact enzyme
and the CBM-deficient versions.792
Product inhibition of cellulases by cellobiose is a point of
interest and noted concern when considering industrial
conversion process conditions and design of experiments, as
detailed in the previous two sections. Conversion rates of
cellulase cocktails tend to drastically decline with increasing
concentrations of glucose and cellobiose during the initial
hydrolysis stage,581 though which enzymes directly contributed
to this effect was not immediately clear. Gruno et al. directly
examined product inhibition for TrCel7A and three Trichoderma
EGs including TrCel5A to delineate contributions from each of
these enzymes to the inhibition effect.611 As described
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Chemical Reviews
previously, the primary cellobiose inhibition effects tend to
derive from processive CBHs. The authors report TrCel5A
hydrolysis of tritiated amorphous cellulose is virtually unaffected
by product inhibition, as was the case for the other EGs in the
study. Comparatively, inhibition of TrCel7A on tritiated BC was
nearly 100-fold higher than any of the EGs. It is likely that the
relatively weak adsorption of TrCel5A with carbohydrate
substrates resulting from the open, shallow binding site cleft
plays a significant role in allowing the cleaved cellobiose product
to rapidly dissociate from the active site preventing any
significant degree of inhibition. Using a completely different
technique of monitoring unmodified cellulose hydrolysis by
calorimetry, Murphy et al. would later corroborate this
finding.615
Gruno et al. also report turnover numbers for the various EGs
on amorphous cellulose. For TrCel5A, amorphous cellulose is
hydrolyzed at a rate of 8.0 ± 0.1 s−1.611 Karlsson et al. previously
reported a kcat of 65 s−1 for TrCel5A on cellopentaose.334 The
disparity is likely not in the actual rate of hydrolysis, but as
Gruno et al. point out, the differences may lie in the means by
which hydrolysis was detected, namely formation of soluble
sugars. In later years, accurate detection of cellulase kinetics has
become a focus area of several research groups around the
world.315,527,795
The means by which EGs and CBHs work together to
deconstruct cellulosic substrates has intrigued the community
for several decades. As described in section 4, the literature
generally points toward an the endo/exo synergism model,
wherein EGs serve to randomly hydrolytically cleave glycosidic
linkages as a sort of preparation for the processive CBHs that
rapidly cleave without dissociation.305 Nidetzky et al. set out to
describe synergistic cellulase action between binary mixtures of
cellulases on filter paper. The authors point out that a key reason
behind many of the inconclusive and contradictory reports was
likely due to insufficient enzyme purity clouding results. Paying
close attention to this fact, the authors reported the
complementary action of both CBH combinations and CBH/
EG combinations. A particularly effective combination was the
addition of TrCel5A with the TrCel7A CBH enhancing overall
activity more than 2-fold. Medve et al. confirm this finding for
hydrolysis on Avicel at 40 °C, but the authors go on to suggest
that TrCel7A and TrCel5A compete for binding sites so as to
negatively affect synergism, with TrCel7A as the more effective
competitor.796 This latter finding suggests selection of enzyme
ratios, erring on the low side of EGs, may effectively minimize
enzyme costs while maintaining endo/exo synergism.797 The
role of EGs in endo/exo synergism, in particular TrCel5A,
continues to be a major research focus today.798−801 As new
approaches for describing cellulase kinetics become available,
models describing endo/exo synergism re-emerge, challenging
the way we think about processive cellulase action and the role
EGs play.557
Thermal stability of an enzyme is essential to industrial
relevance, as increasingly higher temperatures facilitate biomass
decomposition. Intuitively, it would seem that enzymes secreted
from the same native host, such as TrCel6A, TrCel7A, TrCel7B,
and TrCel5A, would have similarly evolved thermal stabilities
given exposure to the same environmental conditions and
stresses. However, this is not necessarily the case as reported in
1992 by Baker et al.802 Using differential scanning calorimetry
and complementary tryptophan fluorescence monitoring, the
authors discovered that thermal deactivation of all four enzymes
is related to thermal unfolding, but that the overall stability of
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TrCel5A is approximately 10 °C higher than either of three
other cellulases (Figure 60). With a Tm of 75 °C, TrCel5A is
Figure 60. Differential scanning microcalorimetry of four T. reesei
cellulases, with concentration of ∼1 mg/mL in cell. In the reproduced
figure, CBHII is TrCel6A, CBH I is TrCel7A, EGI is TrCel7B, and
EGII is TrCel5A. Reprinted with permission from ref 802. Copyright
1992 The Humana Press, Inc.
significantly more tolerant of adverse process conditions than
any of its complements. We note that while a Tm of 75 °C is
significant by comparison, TrCel5A is not considered a
hyperthermophilic enzyme, and thus, protein engineering efforts
tend to focus on further gains in thermal stability as we will
discuss below.
Protein stability in highly alkaline environments is also a
favorable industrial attribute, particularly given the recent focus
on ionic liquid pretreatment of biomass.91,803 Wahlström et al.
investigated the effects of ionic liquids on TrCel5A and
demonstrated that the ionic liquid environment had a markedly
detrimental effect on hydrolysis.804 The authors report the CBM
is particularly susceptible to the effects of ionic liquids as
evidenced by side-by-side comparisons of hydrolysis both with
and without a CBM domain.
8.2.3. H. insolens Cel5A. Few other GH5 fungal cellulases
have been as well-characterized as TrCel5A. Motivated in part
by the industrial applicability, H. insolens EGs have been
catalogued and characterized, though sparingly. Prior to the
initial report of cloning and sequencing,717 HiCel5A (EGII) was
characterized as part of a set of H. insolens cellulases in studies
focused on uncovering stereochemistry, specificity, and kinetics
of several GH families.442,805 The stereochemistry of HiCel5A
was indeterminate from the results of the Schou et al. study.442
However, a similar bacterial EG from family 5 (A at the time)
was shown to follow an inverting stereochemical course in
accordance with earlier reports of E. chrysanthemi EG Z.756 This
account offered yet another clue that GH5 enzymes would all
generally follow this stereochemistry.442 The characterization of
HiCel5A by Schou et al. also suggested the enzyme active site
consists of at least six subsites as affinity for cellohexaitol over
cellopentaitol was significantly higher. This latter finding has yet
to be structurally confirmed.
Several years later, Schülein performed a follow-up study to
identify pH activity sensitivity and ranges as well as to fill in the
gaps regarding stereochemical course and kinetics.499 It had
been noted previously that HiCel5A as well as several other H.
insolens EGs exhibited specificity for CMC; thus, CMC was used
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 Table 11. Summary of Biochemical Characterizations of Fungal GH5 Cellulases
GH 5 substrate for
organism expression host enzyme subfamily pH opt temp opt (°C) opt substrate specificity ref comments
Acremonium sp. Cel5A PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., assay condition 50 °C and pH 5.0; 52% residual
CBS265.95 CMC, xyloglucan, xylan, arabinoxylan, 2010332 activity on CMC after 3 h at 60 °C and pH 5.0
mannan, galactomannan
Aspergillus aculea- Cel5B PASC, Avicel, BC, pretreated corn stover, Vlasenko et al.,
Chemical Reviews

assay condition 50 °C and pH 5.0; 100% residual

tus CMC, xyloglucan, xylan, arabinoxylan, 2010332 activity on CMC after 3 h at 60 °C and pH 5.0
mannan, galactomannan (15% at 70 °C)
Aspergillus f umi- Pichia pastoris Egl2 5 5 50 CMC CMC, filter paper Liu et al., 2011807
gatus
Aspergillus f umi- Pichia pastoris Egl3 5 4 60 CMC CMC, filter paper, Avicel Liu et al., 2011807
gatus
Aspergillus nidu- EglA 5 6.5 50 CMC CMC, Avicel Chikamatsu et al.,
lans 1999808 Bauer et
al., 2006487
Aspergillus nidu- Pichia pastoris EglB 4 52 CMC CMC, cello-oligosaccharides Bauer et al., 2006487
lans
Aspergillus niger Pichia pastoris EglB 5 4 70 CMC barley β-glucan, locust bean gum, cellobiose, Li et al., 2012809
CMC, laminarin
Basidiomycete Cel5A 60 PASC PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., assay conditions pH 5.0; 103% residual activity on
CBS495.95 CMC, xyloglucan, xylan, arabinoxylan, 2010332 CMC after 3 h at 40 °C and pH 5.0 (7% at 60 °C
mannan, galactomannan and 2% at 80 °C)
Basidiomycete Cel5B 60 PASC PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., assay conditions pH 5.0; 28% residual activity on
CBS495.95 CMC, xyloglucan, xylan, arabinoxylan, 2010332 CMC after 3 h at 60 °C (5% at 80 °C)
mannan, galactomannan
Daldinia es- EG 6 70 CMC filter paper, Avicel, CMC Karnchanatat et al., activity stimulated by select divalent ions

1394
chscholzii (Eh- 2008810
renb.:Fr.)
Dictyoglomus Escherichia coli Cel5H 5 50−85 CMC CMC Shi et al., 2013811 maintains 78% activity at 4 M NaCl; chimerical
thermophilum CBMs moderately improve specific activity
toward CMC
Fomitopsis palust- EG47 CMC CMC, pNPC, Avicel Yoon et al., 2007812
ris
Fomitopsis pinico- EG 5 60 CMC CMC, Glc4/Glc5 Yoon et al., 2008813
la
Fusarium verticil- E2 5 80 CMC CMC, barley β-glucan, locust bean gum, de Almeida et al.,
lioides xylan, filter paper, cello-oligosaccharides 2013814
Gloeophyllum tra- Cel5A 5 CMC, pNPC, xylan, PASC, Avicel Cohen et al., suggest GtCel5A is a processive EG due to
beum 2005815 generation of cellobiose from Avicel
Gloeophyllum tra- Pichia pastoris Cel5B 5 3.5 30−70 CMC CMC, filter paper Kim et al., 2012816
beum
Humicola grisea Aspergillus ory- Egl2 5 5 75 CMC CMC, Avicel, xylan, cellobiose, Takashima et al.,
zae Glc3/Glc4/Glc5/Glc6 1997817
Myceliophthora Cel5A 5 PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., assay condition 50 °C and pH 5.0; 50% residual
thermophila CMC, xyloglucan, xylan, arabinoxylan, 2010332 activity on CMC after 3 h at 70 °C and pH 5.0
mannan, galactomannan
Neocallimastix Escherichia coli CelA 4 8.5 40 CMC CMC, Avicel, xylan, lichenan Fujino et al., 1998818
f rontalis
MCH3
Neurospora crassa GH5-1 5 CMC, Avicel Sun et al., 2011819
Orpinomyces joy- CelB2 4 6.6 45 CMC CMC, barley β-glucan, lichenan, pNPC, p- Qiu et al., 2000820
onii nitrophenyl-β-D-cellotrioside
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 Table 11. continued
GH 5 substrate for
organism expression host enzyme subfamily pH opt temp opt (°C) opt substrate specificity ref comments
Orpinomyces joy- CelB29 4 5.8 50 CMC CMC, barley β-glucan, lichenan, pNPC, p- Qiu et al., 2000820
onii nitrophenyl-β-D-cellotrioside
Penicillium brasi- Aspergillus ory- Cel5C 5 4 70 CMC CMC, Avicel Krogh et al., 2009821
lianum zae
Chemical Reviews

Penicillium canes- Escherichia coli 3.4 60 CMC Chulkin et al.,

cens 2009822
Penicillium de- Saccharomyces Cel5A 5 4 60 CMC CMC, barley β-glucan, PASC Wei et al., 2010509 suggest PdCel5A may be processive
cumbens cerevisiae
823
Penicillium de- Pichia pastoris Cel5C 4 4.8 40−50 konjac gluco- glucomannan, tamarind seed gum, CMC, Liu et al., 2013 sequence includes an Ig-like domain
cumbens mannan barley β-glucan
Penicillium echi- Pichia pastoris Egl1 5 5.0−9.0 60 CMC CMC, filter paper, short oligosaccharides Rubini et al., strongly stimulated by calcium
nulatum 2009824
Penicillium janthi- Saccharomyces EGII 5 CMC, hydroxyethylcellulose, barley Mernitz et al.,
nellum cerevisiae β-glucan, lichenan 1996825
Penicillium pino- EG 5 70 CMC CMC, Avicel, cellulose, lichenan, laminarin, Jeya et al., 2010826
philum xylan, Glc4/Glc5
KMJ601
Phanerochaete EG38/Cel5A 5 CMC, Avicel, p-nitrophenyl-β-D-cellotrioside Uzcategui et al.,
chrysosporium 1991827
Phanerochaete EG44/Cel5B CMC, Avicel, p-nitrophenyl-β-D-cellotrioside Uzcategui, 1991827
chrysosporium
Phialophora sp. Pichia pastoris EgG5 4.0−5.0 70 CMC CMC, barley β-glucan, galactoglucomannan, Zhao et al., 2012828 retained 40% activity at pH 2.0
G5 filter paper, Avicel

1395
Piromyces equi Cel5A 4 5.1 45 CMC CMC, barley β-glucan, lichenan, galacto- Eberhardt et al., characterization of CD only; narrow pH optimum
mannan, pNPC, xylan 2000829 range and relatively thermally unstable
Piromyces rhizin- Escherichia coli EglA 4 5.5 50 CMC CMC, pNPC, barley β-glucan, lichenan, Liu et al., 2001776 deletion of the N-terminal linker significantly
f lata xylan, Avicel, filter paper affects thermal stability
Piromyces rhizin- Escherichia coli EglA 4 6 50 CMC CMC, pNPC, barley β-glucan, lichenan, Tsai et al., 2003724 characterization of CD only
f lata xylan, Avicel, filter paper
Talaromyces β-glucanase 4.8 80 barley β-glucan barley β-glucan, lichenan Murray et al., strict preference for mixed linkages, markedly
emersonii CBS 2001830 thermostable
814.70
Thermoascus aur- Cel5A 5 4.0−4.4 70−80 CMC CMC, barley β-glucan, lichenan Parry et al., 2002780 putatively exhibits 5 binding subsites
antiacus
Thermoascus aur- Saccharomyces Cel5A 5 6 70 CMC CMC Hong et al., 2003831
antiacus cerevisiae
IFO9748
Thielavia terrestris Cel5A PASC, Avicel, BC, CMC, mannan, galacto- Vlasenko et al.,
mannan 2010332
Trametes hirsuta Aspergillus ory- Eg-1 5 5 50 CMC CMC, PASC, Avicel, kraft pulp Nozaki et al., long linker may aid in moderate activity toward
zae 2007832 Avicel
Trichoderma sp. Escherichia coli C4endoII 5 5 50 CMC CMC, Avicel Sul et al., 2004833
C-4
Trichoderma ree- Saccharomyces Cel5A 5 4.8 CMC-Na CMC-Na, Avicel, ball-milled cellulose, PASC Qin et al., 2008324 assay condition 50 °C
sei cerevisiae
Trichoderma vir- Saccharomyces EGVIII 5 6 60 CMC CMC, cellobiose, Glc3, Avicel Huang et al.,
ide cerevisiae 2009834
Volvariella volva- Pichia pastoris EG1 5 7.5 55 CMC CMC, PASC, filter paper Ding et al., 2001835
cea
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Chemical Reviews
to determine pH activity profiles. HiCel5A exhibited one of the
broadest pH activity profiles out of the seven H. insolens
cellulases examined maintaining greater than 60% activity over a
pH range 5.5−9. At the optimal pH of 7.5, HiCel5A exhibits a
turnover rate of 8 s−1. Schülein also confirmed HiCel5A acts via
the inverting stereochemical mechanism in this study.499
HiCel5A shares nearly 50% identity with TrCel5A, which is a
striking degree of similarity given the relatively low homology of
GH5s in general. Both of these fungal cellulases belong to
subfamily 5 and exhibit an N-terminal CBM. Schülein was the
first to describe the existence of the HiCel5A CBM explicitly in
the literature, though the sequence had been annotated as
such.499,717 Schülein also concludes that on the basis of the
sequence homology, HiCel5A very likely folds according to the
canonical (β/α)8 motif of GH5s.
The relative effectiveness of CMC hydrolysis by a handful of
industrially relevant EGs was more recently examined.806
Karlsson et al. concluded that HiCel5A was the most active
hydrolyzer of the family 5, 7, 12, and 45 EGs examined alongside
TrCel7B.806 The authors also showed that HiCel5A was one of
the least inhibited enzymes in terms of accepting hydroxyl
substitutions on the CMC substrate. This suggests HiCel5A
may be a bit more forgiving in acceptance of nonideal substrates,
which is ideal in terms of cellulose deconstruction but not for
molecular probe applications.
8.2.4. Other Fungal GH5s. Vlasenko et al. recently
undertook a broad study of EG substrate specificity to identify
family dependent specificities.332 The specificity of five fungal
GH5 cellulases (Aspergillus aculeatus Cel5B, Basidiomycete
CBS494.95 Cel5A, Basidiomycete CBS495.95 Cel5B, Myce-
liophthora thermophilia Cel5A, and Thielavia terrestris Cel5A)
against a variety of substrates was examined, including p-
nitrophenyl substrates, CMC, PASC, Avicel, BC, pretreated
corn stover, xyloglucan, wheat arabinoxylan, β-1,4-mannan, and
galactomannan. Unsurprisingly, family 5 EGs appeared to
exhibit a greater preference for PASC than for the other
polymeric cellulose substrates. The EGs were also incapable of
cleaving p-nitrophenyl substrates, potentially because binding of
the small substrates to the long clefts (seven putative subsites) is
too weak to induce activity. In comparing CMC and PASC
activity levels across all the EGs, Vlasenko et al. suggested that
the GH5 EGs appear to be more accommodating of O-
substitution by methyl groups and rely less on hydrogen
bonding of the substrate for hydrolysis in comparison to family
6, 7, 9, 12, and 45 EGs, a finding that lines up well with those of
Karlsson et al.806 All of the GH5 EGs known to have celluloytic
activity were also remarkably effective at mannan degradation. It
has been hypothesized that the GH5 family has resulted from
divergence of a common ancestral gene,730,731 and this
comprehensive specificity study provides evidence pointing
toward divergent evolution where the ancestral gene exhibited
mannanase activity.
Many additional characterizations of optimum pH and
temperature as well as substrate specificity studies accompany-
ing gene cloning and sequencing efforts have been conducted.
These results are summarized in Table 11.
8.3. Protein Engineering
As with the catalytic mechanism, many of the noted successes in
engineering GH5 cellulases pertain to bacterial GH5 EGs.
Briefly discussing some of the more pertinent advances from
bacterial representatives, we focus on published approaches to
engineering specificity, activity, and stability in GH5s. These
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Review
enhancements have been attained through a variety of
approaches including host engineering, heterologous expression,
and site mutagenesis efforts.
For the most part, efforts associated with GH5 protein
engineering have targeted thermal stability improvements in
industrially relevant cellulases. A wealth of data surrounding
thermophilic and hyperthermophilic GH5 family members
exists by which analogies can be made. To date, both rational
design and directed evolution techniques have demonstrated
moderate success.752,777,778,836,837 Recently, Liu et al. used
directed evolution on a Clostridium phytofermentans GH5 to
improve the half-life by 92% at 60 °C.778 The gain was made
through identifying a triple point mutation, though the means by
which this cluster contributed to stability was not described. As
part of this same study, the authors also investigated effects of
CBMs on GH5 activity noting that removal of native CBMs
from the bacterial EG clearly had a detrimental effect on activity
toward both regenerated cellulose and Avicel.778 Chimerical
CBM and Ig domain engineering frequently negatively affected
activity as well though. A similar approach toward engineering
an uncultured bacterial GH5 EG resulted in a 7-fold increase in
thermal stability with the final enzyme having six mutations and
an appended non-native family 6 CBM.836 This latter CBM-
variant GH5 EG exhibited improved activity toward Avicel. On
the basis of this small sample of directed evolution/chimera
engineering studies one can conclude directed evolution may be
a helpful first approach toward uncovering locations contribu-
ting to thermal stability. However, the appending of non-native
CBMs to the variant does not always result in hydrolytic gains
toward increasingly crystalline cellulose substrates.
Strategic implementation of disulfide bonds has also been
shown to result in thermal stability gains in bacterial GH5
cellulases. Badieyan et al. performed MD simulations on 12
GH5 cellulases to develop a rules-based approach to engineering
stability.752 Correlations with several protein structure dynamic
factors were developed to identify which were more likely
predictors of overall thermal stability. The authors confirmed a
positive correlation between protein flexibility and optimum
activity temperature existed within the subset of GH5s (Figure
61). Using this information, Badieyan et al. identified a
particularly thermally susceptible region of C. thermocellum
CenC. This happened to be the large subdomain insertion
between the α6 helix and β6 strand shown in Figure 53. A new
disulfide bond was introduced in this location with the intent of
stabilizing the protein without affecting activity. Assays of the
wild-type and disulfide-linked mutant with pNPC suggested the
disulfide bridge consistently resulted in improved activities at
elevated temperatures. The Tm of C. thermocellum CenC was
improved by approximately 4 °C with the additional disulfide
bond. As has been noted previously in the literature,675 the
mutation actually had little effect on overall protein fluctuations,
but rather reduced flexibility was sequestered to the subdomain
region. This latter observation likely accounts for the maintained
activity with the addition of the disulfide bond.
Hyperglycosylation of fungal GH5 cellulases by heterologous
expression in yeast has been shown to positively impact thermal
stability. In two separate studies, TrCel5A was heterologously
expressed in S. cerevisiae and P. pastoris.779,838 In each case, the
half-life of TrCel5A at 70 °C was at least doubled as a result of
the hyperglycosylation. Both Qin et al. and Samanta et al. note
that hyperglycosylation did not detrimentally affect specific
activity on CMC, as is sometimes a concern when non-native
glycans block substrate binding.779,838 This approach to
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Chemical Reviews
Figure 61. Using MD simulation of a large set of GH5 EGs from
hyperthermophilic, thermophilic, mesophilic, and psychrophilic organ-
isms, Badieyan et al. illustrated a correlation between protein flexibility,
as measured by root-mean-square deviation (RMSD), and the optimal
activity temperature (OAT) of the enzyme. The authors performed 10
ns MD simulations of each enzyme at three different temperatures. The
correlation appears to be linear. Reprinted with permission from ref
752. Copyright 2011 Wiley Periodicals, Inc.
improved thermal stability is inherently dependent on the
location of readily glycosylated serine and threonine residues at
the surface. Thus, it is unlikely that this is a universal approach to
engineering thermal stability in fungal GH5 cellulases.
Individual enzymes will have to be characterized to ensure
specific activity is not adversely affected in each case, and the
extent to which hyperglycosylation aids in stability may vary.
Stability of TrCel5A at extreme pH values is also a motivator
of several protein engineering studies, the alteration of which
would enable harsher alkaline biomass pretreatment conditions,
a pretreatment option under intense development cur-
rently.839−842 Wang et al. describe the use of directed evolution
to increase the pH optimum of TrCel5A.843 The approach
identified a surface exposed asparagine, Asn321, that had been
substituted by a threonine shifting the optimal pH of TrCel5A
from 4.8 to 5.4. Further site-directed mutagenesis at this site,
N231H, broadened the optimal range to pH 6.0. The authors
conclude this conserved residue is a key determinant of TrCel5A
pH stability. Thus, this mechanism of improved alkaline-
resistance may similarly apply to other homologous GH5s. Qin
et al. later applied the same approach to identify additional
residues contributing to the specific pH profile of TrCel5A. In
this latter study, a series of charged, surface exposed residues
were again shown to contribute to the pH optimum.324 Qin et
al. tested several single point asparagine variants and three
cluster variants, one of which interestingly included a substrate
binding aromatic residue. The alkaline resistivity was improved
to a maximum pH 6.2. The key takeaway message from both of
these studies appears to be that charged residues at the surface of
TrCel5A significantly affect pH stability. Notably, the gains in
TrCel5A pH stability at increasingly higher pH do not yet reach
those of alkaline levels, and significant surface residue
remodeling is likely necessary to achieve such a goal.
Site-directed mutagenesis has also been successfully imple-
mented to alter substrate specificity and activity in fungal GH5s.
After examining conserved residues in several GH5 sequences,
Wang et al. identified several key residues putatively involved
with substrate binding and catalysis in Macrophomina phaseolina
Egl1.844 The wild-type enzyme was capable of hydrolyzing both
CMC and cellopentaose, the shortest cello-oligomer able to be
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Review
hydrolyzed. Mutation of most of the identified residues
abolished activity on both CMC and cellopentaose. However,
the authors noted that one of the conserved aspartate residues
upon mutation to alanine was unable to hydrolyze cellopen-
taose, but that activity on CMC was maintained. A follow-up
assay indicated the D232A M. phaseolina Egl1 variant was
capable of hydrolyzing cellohexaose. The results suggest that
substrate specificity in GH5 cellulases can be modified through a
single point mutation, potentially in the same fashion as the
mutated aspartate was a conserved residue. Nevertheless, the
benefit of increasing minimum EG substrate size with respect to
industrial applications is unclear.
Perhaps one of the more well-known instances of GH5
engineering is the significant activity improvement from a single
active site mutation in A. cellulolyticus Cel5A.845 Baker et al.
mutated a tryptophan implicated in product inhibition (Y245G)
to alleviate stacking interactions slowing the rate at which the
cellobiose product leaves the active site. The improvement in
activity was 40% over wild-type as a result of the staggering
1480% reduction in affinity of the active site for cellobiose, as
measured by the inhibition constant. Unfortunately given that
this residue is not conserved either in type or similarity, it is
difficult to directly define a lesson that may be applied in
engineering fungal GH5s. In fact, TaCel5A already displays a
glycine in the same location suggesting product inhibition in
TaCel5A may have been addressed through natural evolutionary
changes. TrCel5A displays an asparagine and may similarly be
unaffected by product inhibition at this site.
Finally, heterologous hosts capable of rapidly producing
GH5s demonstrating near native activity are particularly useful
for screening enzymes in the search for novel activities and
properties. Nakazawa et al. describe successful recombinant
expression of the key T. reesei EGs, Cel7B, Cel5A, and
Cel12A.846 The authors report the first description of a
prokaryotic host (E. coli) expressing all three EGs from T.
reesei. The authors characterize the activity, stability, and
specificity of each of the recombinant EGs, as they believe
demonstration of properties similar those of the natively
expressed EGs represents a potentially advantageous oppor-
tunity to use the E. coli host in high-throughput mutagenesis
studies. The recombinant TrCel5A CD, the only GH5 of the
three EGs, behaved remarkably similarly to TrCel5A expressed
in T. reesei. The TrCel5A CD, targeting only β-1,4 glycosidic
linkages in model substrates, maintained broad pH range of
4.5−6, with an optimum pH of 5.5 at 50 °C. The enzyme also
maintained stability at 50 °C for 60 min, though activity
dropped by 60% with a 10 °C increase. In general, this study
offers a promising alternative host for high-throughput
expression of TrCel5A mutants.
8.4. Conclusions
GH5 enzymes represent one of the larger and more diverse
groups of GHs with over 20 different hydrolytic modes of action
and at least 51 different phylogenetic subfamilies. Though
relatively few, the fungal GH5 cellulases have been well-
characterized; the industrial relevance of these cellulases is
paramount as this family contributes over 50% of fungal EG
action in the T. reesei secretome.742 Structural and biochemical
characterization efforts over the years have successfully
elucidated the following features of GH5 cellulases:
(1) All of the fungal GH5 cellulases discovered to date are
EGs.
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Chemical Reviews Review

Table 12. Reported GH12 Crystal Structures

source and original name in primary citation PDB code resolution (Å) brief highlights ref
Archaeal Structure
Pyrococcus f uriosus DSM3638 Cel12A 3VGI 1.07 first archaea GH12 structure 874
Bacterial Structures
Bacillus licheniformis DSM13 BlXG12 2JEM 1.78 apo structure 862
2JEN 1.40 ligand complex structure 862
Rhodothermus marinus ITI378 Cel12A 1HOB 1.80 apo structure, first structure of a thermostable GH12 enzyme 875
2BW8 1.54 apo structure 884
2BWA 1.68 bound cellotriose 884
2BWC 2.15 bound cellotetraose 884
3B7M 2.10 thermostable variant enzyme 884
Streptomyces lividans 1376 CelB 1NLR 1.75 first GH12 structure 871
2NLR 1.20 first GH12 ligand complex structure 872
Streptomyces sp. 11AG8 Cel12A 1OA4 1.50 structure of a thermophilic GH12 enzyme 876
Thermotoga maritime MSB8 Cel12A 3AMH 2.09 wild-type apo structure 877
3AMM 1.98 wild-type soaked with cellotetraose 877
3AMN 1.47 E134C variant soaked with cellobiose 877
3AMP 1.78 E134C variant soaked with cellotetraose 877
3AMQ 1.80 E134C variant cocrystallized with cellobiose 877
3O7O 2.41 891
3VHN 2.50 bound cellobiose ligand 887
3VHO 1.93 Y61GG insertion 887
Fungal Structures
Aspergillus aculeatus KSM510 Xeg1 3VL8 1.90 878
3VL9 1.20 bound xyloglucan 878
Aspergillus niger CBS 120.49 EglA 1KS4 2.50 palladium complex 879
1KS5 2.10 879
Humicola grisea ATTC 22081 Cel12A 1OLR 1.20 apo structure 880
1UU4 1.49 cellobiose ligand 208
1UU5 1.70 cellotetraose ligand with mixed linkages binding from −4 to −1 subsites 208
1UU6 1.40 cellotetraose binding from −2 to +2 subsites by cellopentaose soak 208
1W2U 1.52 thio-oligosaccharide ligand spanning active site 208
Hypocrea schweinitzii ATCC 66965 Cel12A 1OA3 1.70 876
Trichoderma harzianum IOC-3844 Cel12A 4H7M 2.07 881
Trichoderma reesei QM9414 Cel12A 1H8V 1.9 first fungal GH12 structure 567
1OA2 1.5 A35V variant 876
1OLQ 1.7 P210C variant 876

(2) GH5 cellulases exhibit the ubiquitous TIM barrel fold.
The generality of this structure allows for significant
variation of the loops connecting the major structural
elements and likely accounts for the wide variety of
observed substrate specificities, thermal stabilities, and pH
optima.
(3) Relatively few GH5 fungal structures have been
determined (only 4 thus far), and much of what is
known regarding mechanistic function has been deter-
mined from bacterial representatives.
(4) The catalytic active site of GH5 enzymes is defined by
two sequence motifs, an Asn-Glu-Pro motif on the β4
strand that includes the catalytic acid and a Glu-Xxx-Gly
motif on the β7 strand (where Xxx is typically an aromatic
residue) that contains the complementary Glu nucleo-
phile. The location of the catalytic acid and base includes
GH5 cellulases in the 4/7 superfamily of enzymes.
(5) Hydrolysis of the glycosidic linkage occurs through a two-
step, retaining mechanism. The glycosylation step
produces a glycosyl-enzyme intermediate resulting in
glycosidic bond cleavage. A water molecule then conducts
nucleophilic attack during the glycosylation step, which
resets the enzyme to its initial catalytic state. Studies have
1398
not yet convincingly determined the rate-limiting step in
GH5 catalysis.
(6) Perhaps as a result of the significant sequence diversity
within the family, GH5 cellulases respond particularly
well to protein engineering efforts. Significant gains in
thermal stability have been made through the introduc-
tion of disulfide bonds and hyperglycosylation with little
adverse impact to activity.
(7) GH5 EGs naturally occur both with and without
appended CBMs. The appendage of this module appears
to be evolutionarily related to the environmental
conditions (i.e., at high-substrate loading conditions,
GH5s do not require CBMs for effective cellulolytic
conversion).
Bacterial GH5s have been instrumental in developing our
understanding of the catalytic mechanisms of cellulases within
this family, and a general understanding of the role these
enzymes play in industrial biomass conversion is clear.
Nevertheless, the role the constellation of loops connecting
the major TIM barrel structural elements plays in determining
specificity and thermal stability remains an outstanding
question. Given the diversity of specificities as well as the
range of thermal and pH tolerances within this family of
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 Table 13. Summary of Biochemical Characterizations of Fungal GH12 Cellulases
temp opt
organism expression host enzyme pH opt (°C) substrate for opt substrate specificity ref comments
Aspergillus aculea- Cmc1/FI- 4.5 50 insoluble cello-oligosac- CMC, PASC, insoluble cello-oligosacchar- Murao et al., 1988892
tus F-50 CMCase charides (average DP ide (av DP 20), Glc4/Glc5/Glc6
20)
Chemical Reviews

Aspergillus aculea- Cel12A xyloglucan, xylan, arabinoxylan, galacto- Vlasenko et al., 2010332
tus mannan
Aspergillus aculea- Cel12B PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., 2010332
tus CMC. xyloglucan, xylan, arabinoxylan
Aspergillus fumiga- Cel12A PASC, Avicel, BC, CMC, xyloglucan Vlasenko et al., 2010332 assay conditions 50 °C and pH 5.0
tus
Aspergillus kawa- Saccharomyces CMCase-I CMC Sakamoto et al., 1995893 assay conditions 30 °C
chii IFO 4308 cerevisiae
Aspergillus niger Escherichia coli EglA 4.5 CMC CMC, xyloglucan, β-glucan Van Den Broeck et al.,
CBS 120.49/ 2001894
N400
Aspergillus niger Kluyveromyces EglA CMC, β-glucan Hasper et al., 2002895 assay conditions 40 °C and pH 4.5
CBS 513.88 lactis
Aspergillus oryzae Aspergillus ory- CelA 5.0 55 CMC CMC Kitamoto et al., 1996489
KBN616 zae
Aspergillus terreus EglD 4.8 50 CMC CMC, Avicel, filter paper Narra et al., 2014896
NIH2624
Chrysosporium EGIII 4.5−6 80 CMC CMC, labeled CMC, filter paper, β-glucan, Emalfrab et al., 2003715
lucknowense Avicel, galacto-mannan
Clonostachys rosea Aspergillus niger EGIII/ o-nitrophenyl-β-D-cello- azurine cross-linked hydroxyethyl cellulose, Goedegebuur et al., Gliocladium roseum Cel12C max Tm of 45.9 °C at pH 5

1399
Cel12C bioside (oNPC) oNPC 2002,853 Sandgren et al.,
2005890
Fomitopsis palustris EG-II/ 3.5−4.0 45−55 CMC CMC, Lichenan, barley β-glucan, Glc6, Shimokawa et al., 2008897 stable up to 55 °C
FFPRI 0507 Cel12 pachyman, laminarin, xyloglucan, gluco-
mannan
Gloeophyllum tra- Aspergillus niger Cel12A 4.5 50 CMC CMC, PASC, filter paper, Glc2/Glc3/Glc4/ Tambor et al., 2012801
beum ATCC Glc5/Glc6
11539
Humicola grisea Aspergillus niger Cel12A 5.5 oNPC azurine cross-linked hydroxyethyl cellulose, Goedegebuur et al., max Tm of 68.6 °C at pH 7, optimum pH of 5.5
ATCC 22081 oNPC 2002,853 Sandgren et al., measured at 40 °C
2005890
Hypocrea schwei- Aspergillus niger Cel12A 5.5 oNPC azurine cross-linked hydroxyethyl cellulose, Goedegebuur et al., max Tm of 49.2 °C at pH 5, optimum pH 5.5 measured
nitzii ATCC oNPC 2002,853 Sandgren et al., at 40 °C
66965 2005890
Myceliophthora Cel12A PASC, Avicel, BC, pretreated corn stover, Vlasenko et al., 2010332 assay conditions 50 °C and pH 5.0
thermophila CMC, xyloglucan, xylan, arabinoxylan
Phanerochaete Cel12A CMC, amorphous cellulose, xylan, mannan, Henriksson et al., 1999898 assay conditions pH 5 and 37 °C
chrysosporium filter paper
K3
Polyporus arcular- Cel3A 4.9 52 CMC CMC, Avicel, PASC, Glc3/Glc4/Glc5/Glc6 Ishihara et al., 2005899
ius 69B-8
Trichoderma reesei Cel12A 5 50 β-glucan CMC, PASC, Avicel, Glc4/Glc5, barley Karlsson et al., 2002334 retained >50% activity at pH 7, and 25% at pH 4; at pH
QM9414 β-glucan, glucomannan, filter paper 5, retained 90% activity at 60 °C, and <10% at 70 °C
Review

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Chemical Reviews Review

Figure 62. In 1997, the first three-dimensional structure of a GH12 enzyme, S. lividans CelB2, was determined at 1.75 Å resolution by Sulzenbacher et
al. (PDB code 1NLR).871 The overall structure fold of SlCelB2, as for all GH12 enzymes with known structures, was found to be a β-sandwich. (A) A
top-down and (B) end-on view of the structure of SlCelB2 (yellow cartoon) is shown aligned with the first fungal GH12 cellulase structure, TrCe12A
(bluewhite cartoon, PDB code 1H8V).567 In each panel, the 2-fluorocellotriosyl ligand from the SlCelB2 structure (PDB code 2NLR) is shown in cyan
stick. The catalytic residue motif is also shown in stick. (C) A close-up of the glycosyl-enzyme intermediate intermediate (PDB code 2NLR) revealed
the SlCelB2 catalytic nucleophile, Glu120, covalently bound to the ligand (yellow and cyan stick).872 A second distinct conformation of the 2-
fluorocellotriosyl ligand was found bound in the crystal structure of SlCelB2 representing the product species from the deglycosylation step of the
double-displacement mechanism (orange sticks). The TrCel12A active site is shown in blue/white sticks for comparison.

enzymes, the GH5 family represents a unique case study for
understanding how protein structure contributes to activity and
stability. Structural and biochemical characterization of a large
set of GH5 enzymes is likely to uncover motifs and mutations
that contribute to stability of of these enzymes.
9. FAMILY 12 GLYCOSIDE HYDROLASES
Family 12 glycoside hydrolase (GH12) enzymes have been
extensively characterized over more than four decades,847 long
before this family of enzymes was classified in the CAZy
database151−153 or even cellulase family H as this group of
enzymes was initially named. Cel12A from T. reesei is most likely
the first GH12 enzyme to be discovered and characterized in any
detail and currently represents the most extensively charac-
terized of all GH12 proteins to date (Table 2). The T. reesei
GH12 enzyme (TrCel12A) was initially called EG III and
occasionally still is by some research groups. TrCel12A was
described in several early studies as a low molecular weight (25
kDa) cellulase. At a total of 218 amino acids, TrCel12A is
relatively small compared to the other identified cellulases from
T. reesei. TrCel12A was found to be only sparsely glycosylated
and is one of the three cellulases from this fungus that does not
exhibit a CBM and linker.319,847−851 The first time that this
“low-molecular-weight” GH12 cellulase from T. reesei was
described in literature was in a paper by Håkansson et al.
from 1973, though the cloning of the TrCel12A gene would not
be reported for another two decades.847,851 Some years later, the
cloning and heterologous expression of TrCel12A in S. cerevisiae
was described.852 The amount of natively expressed TrCel12A
has been reported to be relatively small compared to other
cellulases expressed by this fungus, less than 1% of the total
amount expressed protein.848
Subsequent genome sequencing campaigns following the
discovery of the first GH12 identified a wide range of additional
GH12 gene sequence members, though the family as a whole is
relatively small. At the time of this review, there are 357 GH12
entries in the CAZy database.151−153 Of these 357 entries, 55 are
from Archaea, 187 from Bacteria, 114 from Eukaryota, and 1
entry remains unclassified. In 2002, Goedegebuur et al.
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described the cloning and characterization of 15 fungal GH12
enzymes.853 As part of this study, the authors also proposed a
GH12 subfamily classification structure based on amino acid
sequence alignments of the catalytic cores. Accordingly, GH12s
can roughly be divided into four phylogenetic subfamilies
denoted GH12-1 to GH12-4. Subfamilies GH12-1 and GH12-2
contain fungal representatives; subfamily GH12-3 represents
Streptomyces members, and GH12-4 is a Thermophiles subfamily
containing most the archaeal GH12 enzymes. This initial
subfamily division of GH12 enzymes would later be confirmed
and extended in three additional GH12 characterization
papers.854−856 In the paper from Master et al. in 2008, where
they characterize a GH12 xyloglucanase from A. niger, the
authors state that fungal enzymes with a strong preference for
xyloglucan only can be found in subfamily GH12-2. Vlasenko et
al. later added that the subfamily classifications are largely
undifferentiated in terms of substrate specificity, with EGs and
xyloglucanases frequently sharing subfamily classifications.332
This phenomena likely arises from divergent evolution from a
shared ancestral gene.
The specific function of GH12 enzymes in fungal and
bacterial plant-degrading systems is not well-characterized or
understood, and indeed, many questions remain as to the ”true”
function of GH12 enzymes. Some biochemical data on the
specific activity of these enzymes can be found in the literature,
including studies of the activity on soluble850,857 and insoluble
substrates.334,806,849,855,858,859 A summary of identified activities
of GH12 enzymes is included in Table 13. Several reports have
demonstrated that that GH12 enzymes, in addition to being
endo-β-1,4-glucanases (EC 3.2.1.4), also exhibit activity against
β-1,3/1,4-glucan (EC 3.2.1.73),334,856,860 xyloglucan (EC
3.2.1.151), xylan (EC:3.2.1.8),334,499,850,854,861−865 and lichen-
an.866 However, some GH12s exhibit stronger preferences for
the noncellulosic substrates; the GH12 enzymes from Pyrococcus
f uriosus (EglA)867 and the pathogenic fungus Cochliobolus
carbonum (MLG2)868 are reported to prefer mixed-linked β-
glucans over 1,4-linked polysaccharides. GH12 enzymes have
also been shown to induce extension of plant cell walls, which
has been most extensively characterized in TrCel12A.860,869 For
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Figure 63. Sequence alignment of the six of the GH12 structures reviewed here. Strictly conserved residues are shown in red block, and chemically
similar residues in red text. The blue boxes indicate chemical similarity across a grouping of residues. The secondary structural elements of HgCel12A
are shown above the sequences. The catalytic nucleophile is marked by a yellow star, the proposed catalytic acid/base is marked by a blue star, and the
residue with a supporting role in catalysis is marked by a magenta star. The figure was generated with ESPript (http://espript.ibcp.fr).347

the H. insolens GH12 enzyme, Cel12A, Schülein et al. concluded
that the enzyme’s low activity on celluloses, β-glucan, and other
polysaccharides implied that “the function of this cellulase
remains unclear”, suggesting additional functions remain to be
discovered.499
All GH12 enzymes catalyze the cleavage of the β-1,4-
glycosidic linkages in the various types of β-glucan with a net
retention of the anomeric configuration via a double-displace-
ment mechanism as described in section 4.157 This GH12
catalytic mechanism was first demonstrated in 1997 when
Schülein et al. characterized HiCel12A.499 One year later, Zechel
et al. used ligand labeling studies to identify the catalytic
nucleophile, residue Glu120 of Streptomyces lividans CelB2
(Figure 62).857 The identity of the nucleophile was later also
confirmed for TrCel12A in a mutation study 2000 by Okada et
al.870 The two catalytic residues in all GH12 enzymes are two
invariant carboxylates, Glu116 and Glu200 in TrCel12A, which
are the catalytic nucleophile and acid/base, respectively. These
two catalytic residues are separated a distance of ∼5.5 Å in
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TrCel12A, a distance frequently observed for the nucleophile/
acid−base involved in the retaining mechanism of GHs.444
In addition to the catalytic nucleophile and acid/base residues
(Glu116 and Glu200 in TrCel12A numbering), GH12s involve
a third carboxylate residue (Asp99 in TrCel12A numbering) as
part of a catalytic triad. This catalytic triad was first observed in
GH clan-B enzymes.172,873 A summary of GH12 structures that
are discussed are provided in Table 12.
9.1. Structural Studies
To date, there are a total 34 GH12 structures available in the
PDB, originating from 12 unique GH12 enzymes, one
archaea, 874 five bacterial, 862,871,875−877 and six eukary-
otic.567,876,878−881 Table 12 summarizes the structures solved
and notes the presence or absence of bound ligands. The first 3-
D structure of a GH12 enzyme to be solved was that of the
bacterial S. lividans CelB2 (SlCelB2) at 1.75 Å resolution (PDB
code 1NLR).871 Two years later, the same group presented a
ligand complex structure of SlCelB2 at 1.2 Å resolution
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exhibiting a trapped 2-fluorocellotriosyl ligand covalently bound
to the catalytic nucleophile Glu120 (PDB code 2NLR, Figure
62C).872 This initial series of structures were influential in
determining both the overall fold of the enzyme as well as
uncovering key catalytic components. In 2001, Sandgren et al.
determined the first 3-D structure of a fungal GH12 enzyme,
apo TrCel12A, at 1.9 Å resolution (PDB code 1H8V).567
9.1.1. Overall Structure. GH12 enzymes generally exhibit a
β-sandwich fold (Figure 62A), which together with GH11
enzymes form GH clan-C in the CAZy classification, as
described in more detail below. TrCel12A is a typical structural
example of a GH12 enzyme (Figure 62A) consisting of 15 long
β-strands that fold into two twisted, largely antiparallel β-sheets,
A and B, which pack on top of one another. The convex β-sheet
A consists of six antiparallel strands (A1−6), and the concave β-
sheet B consists of nine largely antiparallel strands (B1−9). The
β-strands in the two β-sheets are numbered consecutively from
1 to 6/9 after their order in the sheets, with β-strands A1/B1
closest to the proposed nonreducing end of the binding cleft
(Figure 62A,B). A single α-helix in the structure packs against
the outer convex surface of β-sheet B, found at the bottom right
in Figure 62A. The enzyme is very compact with dimensions of
approximately 40 Å × 40 Å × 30 Å. Among GH12 enzymes,
there are two highly conserved cysteine residues, Cys4 and
Cys32 in TrCel12A, that form a disulfide bond bridging two β-
strands. Some GH12 enzymes have been observed with
additional cysteine residues,880 but these are not highly
conserved among GH12 protein members. In some proteins,
these nonconserved cysteine residues occur as free cysteines,
and the effect of these free cysteines on catalytic activity and
protein stability has been studied in detail.880
Like many fungal cellulases reported to date, the N-terminal
glutamine of TrCel12A undergoes a cyclization and con-
densation reaction with the amine group of the N-terminus
producing a cyclic pyroglutamate (section 6.4). This cyclization
and condensation of the N-terminus is thought to make the
protein resistant to proteolytic degradation.882 Additionally, as
described at length for GH7 and GH6 cellulases, fungal GH12
enzymes are often found to be glycosylated. A N-acetylglucos-
amine residue is found covalently attached to Asn164 in the
crystal structure of TrCel12A,567 one of two N-glycosylation
amino-acid sequence motifs in the protein, consistent with
previous observations of TrCel12A glycosylation (Figure 63).882
The concave surface of β-sheet B in GH12 enzymes forms a
large crevice in the molecular surface perpendicular to the β-
strand direction of the enzymes, as can be seen in Figure 62A.
This crevice is approximately 35 Å long, 8 Å wide, and 15 Å
deep, and forms the substrate-binding site cleft of GH12
enzymes. It is estimated from the size of the catalyctc cleft of
GH12 enzymes that these have the potential to bind at least six
glycan residues. The crevice of GH12 enzymes contains two
glutamate residues, Glu116 and Glu200 of TrCel12A, which are
invariant throughout GH12. As stated above, it was predicted
that Glu116 was the catalytic nucleophile in TrCel12A, which
was directly confirmed by site-directed mutagenesis studies.870
The bottom of subsites +2 and +3 in the substrate binding
cleft of all known GH12 enzymes appears to be formed by
residues from the “cord” region (Pro129 and Ile130 in
TrCel12A), indicated with a red circle in Figure 64. The proline
ring of the “cord” is sandwiched between two aromatic residues,
Trp120 and Tyr147 in TrCel12A. The cord is structurally well-
conserved between all GH12 enzymes. Given the similar nature
of the folds, GH11 enzymes also exhibit this “cord” region, and
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Figure 64. Cord region of TrCel12A (PDB code 1H8V).567 The red
dashed circle encompasses the cord region. Within this region, four
residues, shown in stick representation, form the basis of the +2 and +3
binding subsites.
for some of the GH11 enzymes, such as T. reesei XynII (a GH11
xylanase), there have been reports that the “cord” region is
flexible upon substrate binding.883 As of yet, there are no signs of
similar conformational flexibility in the GH12 cord region.
9.1.2. GH12 Ligand Complex Structures. The first GH12
ligand complex structure, as mentioned above, was that of
SlCelB2.872 This ligand bound structure of SlCelB2 captured a
2-fluorocellotriosyl ligand, covalently bound to the enzyme,
spanning binding site −3 to −1. Two distinct conformations of
the ligand were observed: one where the ligand was covalently
bound to the catalytic nucleophile, Glu120, forming a glycosyl-
enzyme intermediate,872 and a second form representing the
product species produced after the second reaction step of the
double-displacement mechanism.
The first fungal GH12 ligand complex structure was that of H.
grisea Cel12A (HgCel12A), described in a 2004 paper by
Sandgren et al.208 The authors present a total of four different
HgCel12A ligand complex structures formed by soaking crystals
of the native enzyme with either cellobiose (PDB code 1UU4),
cellotetraose (PDB code 1UU5), cellopentaose (PDB code
1UU6), or a thio-linked cellotetraose derivative (G2SG2, PDB
code 1W2U). The ligands were soaked at pH values preventing
rapid hydrolysis of the ligand and, thus, were able to capture
linkages across the active site of the wild-type enzyme. A
theoretical cellohexaose ligand complex has been proposed from
this wealth of structures (Figure 65, top panel). These
HgCel12A ligand complex structures enabled mapping of the
noncovalent interactions between the enzyme and the glucosyl
chain bound in subsite −4 to +2 of the enzyme (Figure 65) and
shed light on the mechanism and function of GH12 cellulases.
The unhydrolyzed cellopentaose ligand and the G2SG2 cello-
oligomer were both found spanning the active site of the
catalytically active HgCel12A enzyme. In both cases, the
pyranoside bound in subsite −1 displayed a 1S3 skew boat
conformation. After soaking HgCel12A in cellotetraose, a cello-
oligomer was captured bound across subsites −4 to −1 of the
enzyme. Rather surprisingly, this ligand contained a β-1,3-
linkage between the two cellobiose units of the oligomer (PDB
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Figure 65. Cartoon representation of the first fungal GH12 ligand
complex structures from H. grisea Cel12A.208 (A) A theoretical model
ligand complex with a cellohexaose spanning the active site derived
from the 1UU4, 1UU5, and 1UU6 structures is shown. The protein is
shown in translucent magenta cartoon. Aromatic residues lining the
binding cleft as well as catalytic residues are shown in green stick. (B)
The cellobiose ligand (1UU4, yellow stick) was found bound in the +1
to +2 subsites. (C) The cellopentaose ligand (1UU6, gray stick) bound
across the −2 to +2 subsites. The electron density of one pyranose
moiety was unable to be resolved. (D) Soaking in cellotetraose resulted
in a mixed linked cellotetraose ligand (1UU5, cyan stick) spanning
subsites −4 to −1 of the enzyme.
code 1UU5). The β-1,3-linkage between the two cellobiose
units of the complexed structure was proposed to have been
formed via a transglycosylation reaction that most likely had
occurred during the ligand soak of the protein crystals. The
authors also proposed that the close fit of this β-1,3-linkage
ligand found in the HgCel12A liganded structure and the
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binding sites it occupies suggests a mixed β-glucan activity for
this enzyme.
In addition to the SlCelB2 and the HgCel12A ligand complex
structures, various types of bound ligands have been determined
for four other GH12 enzymes revealing detailed information
regarding substrate specificity and mode of action of GH12
enzymes. The first additional GH12 ligand complex structure
determined was that of a thermally stable, bacterial EG
presented in 2006 by Crenell et al.884 The Rhodothermus
marinus Cel12A (RmCel12A) structure was captured in complex
with cellotetraose (PDB code 2BWC) and cellotriose (PDB
code 2BWA) by soaking crystals in a solution containing
cellopentaose prior X-ray data collection. In doing so, the
RmCel12A bound either a cellotetraose or cellotriose ligand in
either one of the two copies of the enzyme in the structure, but
not both. The cellotetraose ligand was found bound in subsite
−3 to +1 and the cellotriose ligand in subsite −3 to −1, all in
equivalent conformations and positions observed in the
HgCel12A complex structure with a bound cellopentaose
molecule (1UU6).
In 2007, Gloster et al. presented a 1.4 Å resolution ligand
complex structure of a GH12 xyloglucanase from Bacillus
licheniformis (BlXG12, PDB code 2JEN).862 The BlXG12 ligand
complex structure was obtained from a crystal of a nucleophilic
variant of this enzyme, E115Q. A crystal of this “dead” variant of
the BiXG12 was soaked with a 20 mM xyloglucan
oligosaccharide mixture prior X-ray data collection. The
BlXG12 ligand complex structure obtained had two structurally
different xylosyl ligands bound in the same substrate-binding
cleft of the enzyme. One ligand spanned the −4 to −1 subsites,
and the second bound to the +1 to +2 subsites. The ligand
bound in −4 to −1 subsites consisted of four β-1,4-glucose
moieties decorated with two α-1,6 linked xylose residues
branching out from the glucose moieties in subsites −3 and
−2. The second ligand, bound in subsite +1 and +2, consisted of
two β-1,4-linked glucose moieties, both decorated with an α-1,6
linked xylose residue. The authors concluded that the lack of
additional decoration of the two xyloglucan-ligands bound in the
BlXG12 complex structure, in addition to the bound α-1,6
xylose residues, indicates that this enzyme has a preference for
“naked” β-1,4-glucan chains over more decorated types of β-1,4-
glucan.862
In a paper by Cheng et al. in 2011,877 four ligand complex
structures of T. maritima Cel12A (TmCel12A) were presented.
These ligand−complex structures were obtained after soaking
crystals of TmCel12A, either wild-type protein or an E134C
variant, in either cellobiose (PDB codes 3AMN and 3AMQ) or
cellotetraose (PDB codes 3AMM and 3AMP). In the two
TmCel12A cellobiose soaked complex structures, two copies of
a cellobiose ligand was found bound in the substrate-binding
cleft. The first one was bound in subsites −2 and −1 and the
second one in subsites +1 and +2. In the two TmCel12A
cellotetraose soaked ligand complex structures, a cellotetraose
molecule was found bound to subsites −4 to −1, and in addition
to this, a cellobiose molecule was also bound in binding sites +1
and +2. It is notable that the glucose molecules occupying
binding site −1 in all four TmCel12A ligand complex structures
presented in the paper displayed an α-anomeric configuration.
9.2. Plant Cell Wall Loosening/Extension Activity by GH12
Enzymes
Some GH12 enzymes have been found to exhibit plant cell wall
loosening and extension capability. This capability has most
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Figure 66. Superimposition of GH clan-C members, TrCel12A and TrXynII. (A) Top-down and (B) end-on views of GH12 TrCel12A (blue/white
cartoon, PDB 1H8V)567 structurally aligned with GH11 TrXynII (orange cartoon, PDB 1ENX)885 reveal high structural similarity between the two
families despite very low sequence similarity. The catalytic triads are shown for each structure in stick representation.
been extensively characterized for TrCe12A and was described
in great detail in an extensive paper by Yuan et al. in 2001.860
The same group revisited the topic in a subsequent manuscript
in 2012.869 In the first of these two studies, Yuan et al.
demonstrated that TrCe12A has the ability to induce extension
of heat-inactivated type I cell wall from cucumber (Cucumis
sativus) and wheat (Triticum aestivum) type II cell walls.860 The
authors show that TrCel12A hydrolyzes an important structural
polysaccharide of the cell wall, thereby leading to a significant
structural weakening of the wall. The weakening was most
readily detected as an increase in wall plasticity and elasticity.
Because TrCel12A readily hydrolyzes wall glucans such as
xyloglucan and β-1,3/β-1,4-glucans but not xylans, mannans, or
galactans, the authors conclude that this type of glucan
hydrolysis is the probable mechanism for the TrCel12A effects
on the wall. The overall conclusion of these two papers is that
TrCel12A has the capability to modify both type I and II plant
cell walls, but substantial extension of these is found only in type
I cell walls.869
9.3. GH Clan-C: Structure and Sequence Comparison
The GH clan-C, including GH11 and GH12, consists of 4 major
groups of sequences that correspond to the bacterial and fungal
members in each of the two GH families. Despite low sequence
identity between the two different subgroups (bacterial and
fungal) within each one of these two GH families and the virtual
absence of identity between the two GH families forming GH
clan-C, the overall 3-D structures for all GH clan-C members
exhibit remarkable similarity. Figure 66 illustrates the super-
imposition of a GH12, TrCel12A, with a GH11, TrXynII. The
primary structural difference between the two GH clan-C
members is the absence of the first two β-strands, A1 and B1,
TrXynII. This is true of more than half of GH11s. Nevertheless,
a structural-based sequence alignment of GH clan-C members,
extended to include sequences for which the structure is
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unknown, reveals that only three amino acids are completely
conserved across clan GH-C. Two of the conserved amino acids
correspond to the active site nucleophile and the catalytic acid/
base (Glu 116, and Glu 200, respectively, in TrCel12A), both of
which are essential to catalysis. The third conserved residue is a
valine residue (Val160 in TrCel12A) that is located on the inner
concave β-sheet.
9.4. Enzyme Discovery and Engineering
GH12 enzymes are the most commonly used enzyme for
enzymatic stonewashing of cloth and enzyme additive in
washing powders. These enzymes are produced and sold in
bulk quantities by many enzyme producing companies. Due to
the commercial interest in utilizing GH12 enzymes as
biocatalysts in various biotechnological applications, such as
the ones mentioned above, there has been a relatively strong
market drive to initiate research programs aimed toward
discovery of new, naturally occurring GH12s with suitable
properties for these applications and pro-
cesses.853,859,862,874−877,886 Specifically, many of the processes
for which the GH12 enzymes are suitable biocatalysts are carried
out at elevated reaction temperatures, 80−100 °C, and under
very basic reaction conditions, pH 9 or higher. A direct outcome
of these sometimes very harsh application conditions has been
that two of the most important goals for GH12 research
programs have been to either (1) identify new GH12 enzymes
in nature with increased thermal stability with respect to
catalytic activity and/or better performance properties at basic
or nonphysiological pH conditions,853,859,874−877,880,886−889 or
(2) find ways to change/improve these biophysical properties of
a GH12 enzyme of interest.
Two of the first thermostable GH12 enzymes to be cloned
and characterized in some detail were the two endo-cellulases
from Thermotoga neapolitana, TnCelA and TnCelB. Book et al.
presented the results from the study of these two enzymes in
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Figure 67. Cartoon representation of TrCel12A showing conformational changes close to residue 35 upon mutating this residue from an alanine to a
valine. The point mutation stabilizes the variant enzyme (blue/white cartoon, PDB 1OA2) by 7.7 °C in comparison with the wild-type enzyme (wheat
cartoon, PDB 1H8V). (A) A close-up of the site of the A35 V mutation and (B) overall enzyme structure show the position of residue 35 relative to its
surroundings. A red dashed circle in part B indicates the “cord” region of GH12 enzymes.
1998.864 The authors show that the E. coli expressed TnCelA
and TnCelB enzymes have optimal activities at 95 °C at a pH of
6.0 and 106 °C at a pH of 6.0−6.6, respectively. No conclusions
were drawn regarding what biophysical properties might render
these two so much more thermostable by comparison to other
previously studied GH12 enzymes.
One year later, Bauer et al. described the characterization of a
hyper-thermostable GH12 EG from the thermophilic archaeon
P. f uriosusis, Pf EglA.867 The authors show that Pf EglA has an
optimal activity temperature of 100 °C at a pH of 6.0 and a half-
life of 40 h at 95 °C. Differential scanning calorimetry was used
to determine the denaturing temperature of 112 °C. The
ultrahigh resolution (0.8 Å) structure of Pf EglA was later
described by Kim et al.874 Here, the authors use structural based
evidence to conclude that the hyper-thermostability of Pf EglA
can be explained in part by a calcium ion bound in a Ca2+
binding DxDxDG loop motif located in a loop connecting two
β-strands at one end of the binding cleft of Pf EglA. The authors
also show that if the bound calcium ion is removed from the
enzyme by EDTA treatment prior heat treatment of the enzyme,
a dramatic decrease in residual activity of Pf EglA after heat
treatment occurs.
The first crystal structure of R. marinus Cel12A (RmCel12A),
a highly thermostable GH12, helped reveal ion pairs and
charged surface residues as an additional mechanism of
stabilization.875 The authors show that RmCel12A retains 75%
of its initial catalytic activity after 8 h incubation at 90 °C.
Structural insight aided the authors in describing the origins of
thermostability in RmCel12A; a comparatively large number of
ion pairs in combination with other features such as stabilization
of an active site loop, and an increase in the number of polar
amino acids on the surface of the protein, were reported as
responsible for stabilization.
In additional to identifying new GH12 enzymes from nature
with better overall performance at elevated process temper-
atures and nonphysiological pH values, extensive research
programs have also been initiated with the specific goal to
identify amino acids in the target enzymes that are important for
the thermal stability of the molecule or pH optimum, with
respect to both overall stability and catalytic activity at elevated
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temperatures, and thus identify genetic engineering tools for the
optimization of these properties. Many of the protein
engineering strategies for GH12s have been described in a
2005 review,890 and thus, we only briefly point to very successful
efforts as examples. Such examples of the outcome of such
protein stability engineering campaigns of GH12 enzymes are
presented in the studies by Sandgren et al.,876,880 Nakazava et
al.,888 and Cheng et al.887 In the first of these studies, Sandgren
et al. demonstrate that single amino acid substitutions can be
immensely valuable in improving process relevant properties of
GH12.876 The authors performed point mutagenesis studies
alongside structural comparisons of homologous GH12
enzymes that were less or more thermally stable to suggest
the A35 V substitution in TrCel12A. This point mutation
successfully increased the Tm of the enzyme by 7.7 °C over wild-
type (Figure 67). On the basis of structural evidence, the authors
concluded that the thermal unfolding of TrCel12A most likely
starts at the position of the identified thermal stabilizing A35 V
mutation (Figure 67B).
Some attempts to engineer the pH optimum of GH12
enzymes have also been carried out. Unfortunately, these have
not been as successful as recent thermal stability engineering
efforts. Thus far, the most efficient strategy for identifying GH12
enzymes active at a nonphysiological pH has been to screen for
naturally occurring enzymes with altered pH optimum with
respect to catalytic activity (e.g., in various extreme environ-
ments often found in soda lakes). This type of natural habitat
screening for enzymes with altered pH optimum has been
described by van Solingen et al.886 and Goedegbuur et al.853
Huang et al. also described the characterization of a GH12
enzyme from the thermoacidophilic archaeon Sulfolobus
solfataricus;859 this organism secretes an enzyme having a
remarkable pH optimum of 1.8.
9.5. Conclusions
GH12 enzymes are industrially relevant proteins given that
some of these are important components in commercial
detergent and conditioning formulations. Despite that GH12
cellulases are important components in industrial applications
and reasonably well-characterized structurally, the true function
for these enzymes in the fungal plant cell wall degradation
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Table 14. Reported GH45 Crystal Structures
source and original name in primary citation PDB code resolution (Å)
Fungal Structures
Humicola insolens EGV (HiCel45A) 1ENG
2ENG
3ENG
4ENG
Humicola grisea Egl3 (HgCel45A) 1HD5
Melanocarpus albomyces Cel45A (MaCel45A) 1L8F
1OA7
1OA9
Mollusk Structure
Mytilus edulis Cel45A (MeCel45A) 1WC2
machinery is not well-understood. Here, we generalize the
findings discussed above for GH12s:
(1) The T. reesei GH12 enzyme TrCel12A is most likely the
first GH12 enzyme to be discovered and characterized in
any detail. TrCel12A was initially called EGIII, was
described in several early studies as a low molecular
weight (25 kDa) cellulose, and is one of the three
cellulases from this fungus that does not exhibit a CBM.
(2) Phylogenetic analysis suggests that GH12s can roughly be
divided into four phylogenetic subfamilies denoted
GH12-1 to GH12-4. Subfamilies GH12-1 and GH12-2
contain fungal representatives. As of yet, specificity has
not been strongly linked to these phylogenetic divisions.
(3) GH12 enzymes are among the smallest carbohydrate
active enzymes, with dimensions of approximately 40 Å ×
40 Å × 30 Å. The small size may aid in giving these
enzymes access to small pores in the plant cell walls.
(4) The catalytic domains of GH12 enzymes exhibit a β-
sandwich fold, consisting of 15 long β-strands that fold
into two twisted, largely antiparallel β-sheets, which pack
on top of one another.
(5) The long substrate-binding cleft of the enzyme is formed
by concave surface of the β-sandwich.
(6) The GH12 enzymes exhibit a wide range of cell wall
polysaccharide specificities and catalyze the cleavage of β-
1,4-glycosidic linkages with a net retention of the
anomeric configuration via a double-displacement mech-
anism.
The specific function of GH12 enzymes in fungal and
bacterial plant-degrading systems is not well-characterized or
understood, and indeed, many questions remain as to the “true”
function of GH12 enzymes. Thus, much remains to be
accomplished toward a better and more complete understanding
of these enzymes and their hydrolytic mechanisms. Never-
theless, these enzymes play an important role in both cell wall
deconstruction and generation, suggesting further examination
of enzymes from this interesting GH family is warranted in the
context of industrial applications.
10. FAMILY 45 GLYCOSIDE HYDROLASES
Family 45 glycoside hydrolase (GH45) enzymes share several
common features with GH12 enzymes; they are generally small
by comparison with other GH families, have broad substrate
specificity, and can degrade other cell wall polysaccharides in
addition to cellulose. It is hypothesized that the size of the
enzymes represents an evolutionary advantage, which allows
them to penetrate into smaller pores and cavities and thus gain
Review
brief highlights ref
1.6 first GH45 structure, apo, obsolete 903
1.5 replaces the original 1ENG apo structure 904
1.9 wild-type, cellobiose complex 904
1.9 D10N variant, cellohexaose complex 918
1.66 apo unpublished
1.8 apo 912
2.0 cellobiose complex 914
2.0 apo 914
1.2 apo 917
better access to the substrate. Within GH45, there exist some of
the smallest known cellulases, for example, T. reesei Cel45A
(TrCel45A; also known as T. reesei EG V) consisting of a CD of
only ∼165 amino acid residues. Beyond their size, another
particularly intriguing aspect of GH45s is that they are
structurally and evolutionarily related to expansins, which are
ubiquitous in plants, and swollenins, which are present in some
fungal species, as discussed below.
GH45 (family K in the original classification of cellulases) was
first defined in 1993 by Henrissat and Bairoch when they
extended the GH classification with newly available sequen-
ces.900 At that time, GH45 consisted of only two members, the
bacterial EG EGB from Pseudomonas f luorescens subsp. cellulosa
(now Cellvibrio japonicus) and the fungal EGV from H. insolens
(HiCel45A). The cloning of the bacterial EGB-encoding gene
and characterization of the enzyme was published in 1990 by
Gilbert et al.,901 where they recognized that the 482-residue
enzyme exhibits modular organization. By expression of
truncated variants, it was demonstrated that the catalytic activity
resides in the C-terminal domain composed of ∼250 residues
and that binding to cellulose is nearly abolished when the N-
terminal half is removed. It was found that the enzyme actually
contains two CBMs, an N-terminal ∼100-residue family 2 CBM
followed by a ∼30-residue family 10 CBM. The two CBMs are
separated from each other and from the GH45 catalytic module
by typical serine-rich linkers of ∼50 residues each. Activity was
highest on lichenan and barley β-glucan, successively lower on
CMC and PASC, and very low on crystalline cellulose (Avicel
and filter paper). No activity was detected on xylan, laminarin,
cellobiose, or artificial cellobioside substrates (4-methylumbelli-
feryl-β-D-cellobioside, pNPC).901 This activity profile is typical
for GH45 cellulases.
The other enzyme, HiCel45A, consists of 284 residues and is
also modular, but with the GH45 module (∼210 residues) at the
N-terminus followed by a linker and a C-terminal family 1 CBM.
The preparation and sequence of the enzyme and its properties
for treatment of cotton textiles and other cellulose fibers was
described in a patent 1991 from Novo Nordisk A/S (now
Novozymes), Denmark.902 Novozymes has successfully ex-
ploited the enzyme for textile applications such as depilling,
biopolishing, and denim stonewashing, and the product
Carezyme is of widespread use in laundry washing powders.
They are often called “color-restoration” washing powders to
allude to the color-brightening appearance resulting from the
removal of loose fibrils from the surface of cotton fibers.
HiCel45A is one of the most extensively studied members of
this cellulase family and has served as a reference protein for
GH45. The first three-dimensional structure and identification
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Figure 68. GH45 phylogeny (adapted from Sakamoto and Toyohara).909 Sequences from the Plantae, Mollusk, Fungi, Insect, Protozoa, Nematode,
and Bacteria kingdoms are included and colored according to the legend. The GenBank identifier for each sequence is shown in parentheses. At right,
the vertical bars mark the proposed subfamily classification of each sequence. The Plantae sequences are expansins rather than GH45s and thus are not
included in the subfamily classification scheme. The multiple sequence alignment from which the phylogenetic tree was generated was created using
Clustal Omega.910 Only the CDs of the sequences were included in the alignment. The phylogenetic tree was generated with UGENE using the
neighbor joining method and the Jones-Taylor-Thornton distance matrix model.911 The branch length scale (subtitutions/site) is shown at bottom
left.

of catalytic residues903,904 as well as demonstration of an
inverting reaction mechanism in GH45 were obtained from this
enzyme.442 A summary of GH45 structures discussed is
provided in Table 14.
Other examples of modularity within GH45 enzymes have
been observed in, e.g., Pichia pastoris, Mucor circinelloides, and
Piromyces equi. PpCel45A from P. pastoris GS115 (Komagataella
pastoris GS115) carries no less than five different family 1 CBMs
connected to the C-terminal GH45 CD.905 In M. circinelloides
there are two isoenzymes with identical GH45 CDs and either
one or two family 1 CBMs at the N-terminus, which are
suggested to be produced from the same gene by alternate
splicing.906 The first GH45 enzyme to be described in an
anaerobe, the rumen fungus P. equi, has no CBM, but three
fungal dockerins of ∼40 residues each N-terminally connected
to the GH45 CD.829
Today there are over 300 GH45 entries in the CAZy
database,151−153 almost exclusively from Eukaryota. Only a few
bacterial species are present, such as the shipworm symbiont
Teredinibacter turnerae.907 GH45 genes have been found in
many fungi, within Ascomycota, Basidiomycota, Mucorales, and
Neocallimastigales (P. equi). Otherwise, they seem scattered
1407
among rather diverse organisms, including primitive protists
(Parabasilian symbionts in the hindgut of termites), nematodes
(e.g., the pine wood nematode, Bursaphelenchus xylophilus),
mollusks (e.g., the freshwater snail Ampullaria crossean and the
edible Blue mussel, Mytilus edulis), a narrow group of insects
(Cucujiformia beetles, e.g., the mustard beetle, Phaedon
cochleariae, and the rice weevil, Sitophilus oryzae), and one
arthropod species (the Antarctic springtail, Cryptopygus
antarcticus).
On the basis of phylogenetic analysis, a division of GH45 has
been proposed into three subfamilies, A, B, and C.905,908 Two
main evolutionary lineages can be recognized among GH45s.909
The known bacterial and most fungal enzymes, including
HiCel45A, belong to subfamily A together with insect,
protozoan, and nematode members (Figure 68). Subfamily B,
found in mollusks and a few fungi (e.g., M. edulis Cel45A/
MeCel45A and TrCel45A), and subfamily C, so far only
represented by a few basidiomycete enzymes (e.g., P.
chrysosporium Cel45A/PcCel45A), show higher sequence
similarity with each other and with plant expansins and the
related fungal swollenins than the similarity with subfamily A
(Figure 69).
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Figure 69. Structural-based sequence alignment of four GH45 enzymes: HiCel45A, MeCel45A, TrCel45A, and PcCel45A. HiCel45A belongs to
subfamily A, MeCel45A and TrCel45A belong to subfamily B, and PcCel45A belongs to subfamily C. For comparison, two expansins, a swollenin, a
loosenin, a GH45-like domain, and a lytic transglycosylase are shown. The expansins are Solanum lycopersicum (Tomato) α-expansin, SlEXPA, and Zea
mays (maize) pollen allergen/β-expansin, ZmEXPB1. The swollenin is T. reesei swollenin, TrSWO1. The loosenin is Bjerkandera adusta Loosenin,
BaLOOS. The GH45-like domain is Emericella nidulans/A. nidulans cellulase/allergen, EnCelA, and the lytic transglycosylase is E. coli lytic
transglycosylase, EcMltA. Strictly conserved residues are shown in red block, and chemically similar residues are in red text. The blue boxes indicate
chemical similarity across a grouping of residues. Given the diversity of tertiary structure across this family, a consensus sequence representative of
general structure is not available. Thus, the secondary structural elements of each sequence are marked in the following fashion: α-helices are marked
by magenta boxes across the sequences, β-sheets are marked by bolded letters and numerically identified above the sequence. The catalytic acid is
marked by a yellow star, and residues in a conserved motif near the catalytic acid are marked by magenta stars. The alignment was generated with
ESPript (http://espript.ibcp.fr).347

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Figure 70. Top view (left panel) and end-on view (right panel) of the structure of H. insolens Cel45A D10N mutant crystallized with cellohexaose
(4ENG; gray cartoon). Catalytic residues are indicated (red stick), as well as the “lid-loop” (111−118; blue cartoon and stick), other residues of
interest (blue stick), and the two cellotriose units seen in subsites −4 to −2 and +1 to +3, respectively (cyan stick). The 111−118 loop and Asp114
residue (magenta cartoon) of HiCel45A wild-type in complex with cellobiose (3ENG; pink stick) and the corresponding loop in the M. albomyces
Cel45A structures 1L8F (pale green cartoon) and 1OA7 (pale yellow cartoon) are also shown.

10.1. Structural Studies
To date, there are eight GH45 structures of four different
enzymes available in the Protein Data Bank (Table 14). Three of
these enzymes belong to subfamily A: HiCel45A (2ENG,
3ENG, 4ENG),903,904 H. grisea var. thermoidea (HgCel45A;
1HD5), and M. albomyces (MaCel45A; 1L8F;912 1OA7,
1OA9913,914). The structures of the Humicola enzymes were
obtained without the C-terminal linker-CBM,904,915 whereas
with M. albomyces, the native enzyme could be used since it only
consists of a GH45 module. The CDs of these three enzymes are
very similar. The two Humicola CD sequences differ by only
three amino acids and exhibit ∼75% sequence identity with M.
albomyces.912 The fourth enzyme belongs to subfamily B,
MeCel45A (1WC2). The isolation, characterization, and
sequencing of MeCel45A have been published,916 but the
crystal structure is so far only described in a Doctoral thesis at
Uppsala University.917
10.1.1. Subfamily A. The first GH45 structure, HiCel45A
in the apo state (PDB code: 1ENG/2ENG), was published 1993
by Davies et al. revealing a six-stranded, double-psi β-barrel
domain, a fold shared by multiple protein superfamilies.903,919
Loops connecting the β-strands protrude from opposite sides of
the barrel to form a substrate-binding groove along one face of
the β-barrel. The two catalytic residues Asp121 and Asp10,
identified by site-directed mutagenesis, define the catalytic
center near the middle of the groove, which spans seven subsites
from −4 to +3 (Figure 70). Subsequently, Davies et al.
published two ligand-bound complexes, the wild-type enzyme
with cellobiose and the catalytically deficient mutant, D10N,
crystallized with cellohexaose (PDB code 3ENG and 4ENG,
respectively).904 The cellobiose ligand binds in the +1 and +2
subsites, and in the D10N mutant, two cellotriosyl moieties are
seen, one in subsites +1 to +3 and one in −2 to −4 with subsite
−1 unoccupied (Figure 70). In both structures, the free C4
hydroxyl of the glucosyl unit bound in +1 subsite, corresponding
to a glycosidic oxygen in a cellulose substrate, is hydrogen
1409
bonded to Asp121. This latter observation supports the role of
Asp121 as the catalytic acid. The authors note the surprising lack
of carbohydrate−aromatic stacking interactions between the
substrate and the enzyme, with the exception of Trp18 in the −4
subsite.
The HiCel45A structure appears to exhibit significant
conformational changes upon substrate binding that may be
necessary for induction of catalysis. In the apo structure
(2ENG), the loop formed by residues 111−118 did not exhibit
sufficient electron density to make it possible to build this part of
the structure. However, in the cellobiose (3ENG) and the
cellohexaose (4ENG) complexes, the loop is ordered, but with
significantly different conformations found in the two ligand
complex structures. The cellohexaose complex indicates that
substrate binding induces “lid closure”, which brings a third
aspartate, Asp114, near to the −1 subsite and with the
carboxylate group pointing toward the catalytic center. Asp114
also interacts with OH6 of the +1 sugar, as well as with an
opposing loop across the active site via van der Waals contacts
(with Ile131), and two water-mediated hydrogen bonds (to
Ile131 N and Tyr147 OH) effectively enclose the active site in a
short tunnel at the point of cleavage. Davies et al. also
demonstrated that the activity is essentially abolished in the
D10N and D121N mutants, whereas the kcat is reduced by a
factor of 40 in the D114N mutant.904
Interestingly, the distance between the glucose residues in the
−2 and +1 subsites is about 1 Å longer than expected for either a
relaxed or a distorted glucose residue (such as found in
lysozyme191) in the −1 subsite. The authors speculate that this
might be because the enzyme has evolved to optimally bind to
the transition state and that elongation occurs upon protonation
during the inverting hydrolysis mechanism (Figure 71). Davies
et al. subsequently published a full-length article on further
structural refinement of the native HiCel45A enzyme.918
The structure of HgCel45A was deposited in the PDB in 2000
(1HD5; McAuley, K.E., Wilson, K.S., and Schülein, M.), but a
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Figure 71. Davies et al. illustrated their hypothesis for the catalytic
mechanism of HiCel45A in their 1995 manuscript.904 The conventional
inverting catalytic mechanism is proposed to include elongation of the
glycosidic bond in the transition state (middle panel). This supposition
was supported by structural evidence indicating the glucose rings in the
−2 and +1 binding subsites were 1 Å further apart than known sugar
geometries of the time. Reprinted with permission from ref 904.
Copyright 1995 American Chemical Society.
publication has not been reported on this structure. Given that
only three amino acids differ from the HiCel45A enzyme, it is
not surprising that the overall structure is quite similar.
Two structures of MaCel45A were independently published
by two groups in 2003.912,914 Valjakka and Rouvinen reported
the structure of the native apo enzyme (1L8F).912 Hirvonen and
Figure 72. Difference in architecture between subfamilies A and B of GH45. (A) Structure 4ENG of H. insolens Cel45A in subfamily A. (B) Structure
1WC2 of MeCel45A in subfamily B. (C) Superposition of the two structures reveals high similarity at the β-barrel core, while loop regions differ in
length and structure. The proposed catalytic acid residues (red stick) are shown, as well as the −4 tryptophans, conserved residues around the catalytic
acid, and Asp114 of HinCel45A and the corresponding Asn109 of MeCel45A. The two cellotriose ligands in the 4ENG structure are included in panel
C (cyan stick). The orientation and scale are identical in all three panels.
1410
Review
Papageorgiou reported both the apo enzyme and a cellobiose
complex (1OA9, 1OA7, respectively).914 As expected from the
high sequence similarity with HiCel45A, the structures are quite
similar to less than 1 Å RMSD between the structures in all
cases. In the cellobiose complex structure, the ligand is bound in
the −2 and −3 subsites instead of the +1/+2 subsites found in
HiCel45A.904,914 The bound glucose residues are slightly shifted
relative to the corresponding glucan units in the HiCel45A
cellohexaose complex structure.
10.1.2. Subfamily B. To date, there is only one structure
reported in subfamily B of GH45, that of MeCel45A from the
North Sea blue mussel M. edulis; this enzyme consists of a GH45
catalytic module with 181 amino acids and no other
subdomains.917 There are known fungal representatives of this
GH45 subfamily,151−153 but no structures have been reported
thus far. The M. edulis enzyme was discovered after
homogenization of whole mussels, brief boiling, removal of
the solids, and analysis of the supernatant for cellulase
activity.916 Subsequently, the enzyme was localized to the
crystalline style of the digestive tract. The crystalline style is a
rotating, gelatinaceous rod in the stomach of herbivorous
mollusks, which acts as a mechanical device to grind the food in
a mortar-and-pestle fashion.920 The structure of the native
enzyme isolated from crystalline styles was solved at 1.2 Å
resolution (PDB code: 1WC2).
The MeCel45A enzyme shows only 13% sequence identity
with HiCel45A from subfamily A, reflecting a comparatively
large phylogenetic distance between subfamilies A and B.
Nevertheless, the β-barrels superimpose well with an RMSD of
1.6 Å over 105 matched Cα positions, and the structures are
quite similar around the catalytic center. The catalytic base
(Asp24) and the catalytic acid (Asp132, or Asp10 and Asp121 in
HiCel45A) and residues surrounding the catalytic acid are
preserved in nearly identical positions (Thr20, Tyr22, Ala50,
Ala51, His130 in MeCel45A corresponding to Thr6, Tyr8,
Ala73, Ala74, His119 in HiCel45A) (Figure 72). Furthermore,
Asn109 in MeCel45A occupies a similar position as Asp114 in
the flexible loop of HiCel45A upon “lid closure”, where it may
also serve for binding to OH6 of the +1 glucoside. The
corresponding loop in MeCel45A, which carries Asn109,
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exhibits more protein−protein contacts and is not likely to show
similar flexibility as seen in HiCel45A. In both enzymes, a
tryptophan platform is exposed at subsite −4, although the
residues come from different regions of the sequence (Trp64 in
MeCel45A, Trp18 in HiCel45A) and their side chain indole
planes are tilted by ∼80° relative to each other.
Apart from the β-barrel core and the active center region, the
structures of MeCel45A and HiCel45A are markedly different in
architecture due to differences in length of regions that
interconnect the β-strands of the barrel. Such regions are
extended largely along either one or the other side of the β-
barrel in the two enzymes. In MeCel45A, three elongated loops
along one side extend the surface of the barrel and make up one
wall of the active site (Figure 72B), but all the connections on
the other side are rather short and consequently the cleft is more
open and shallower than in HiCel45A. The substrate binding
cleft of the latter enzyme is primarily formed by loops on the
opposite side compared to MeCel45A. However, HiCel45A has
one extended loop on the other side as well, the flexible “lid”
mentioned above, and the binding groove is more enclosed.
The two distinct patterns of longer and shorter regions
between the strands of the β-barrel also show up in the
alignment of available GH45 sequences, consistent with the
difference in architecture between subfamily A on one hand and
B and C on the other (Figure 69). In addition to showing high
sequence similarity to other GH45s from mollusks, M. edulis
Cel45A is quite similar to GH45s from fungal species such as
Trichoderma strains. For example, TrCel45A exhibits 33%
sequence identity with MeCel45A, and the alignment points
to a similar overall shape with an open and shallow active site
groove.
10.1.3. Subfamily C. Upon publication of the expression
and characterization of a GH45 enzyme from the white-rot
basidiomycete fungus P. chrysosporium, Igarashi et al. suggested
that the protein should be classified into a new subfamily C in
GH45.908 The PcCel45A enzyme consists of a 180-residue
GH45 module without linker-CBM. No structure for GH45
subfamily C cellulases has been published to date. However,
given their sequence similarity to subfamily B, it is likely that
they will exhibit structures that are more similar to those from
subfamily B than A. The sequence identity is 21% between
PcCel45A and MeCel45A and from the length of loop regions, it
can be predicted that PcCel45A should have a shallow groove
similar to that of MeCel45A (Figure 69).
Quite interestingly, while the proposed catalytic acid and its
surroundings seem to be well-preserved, PcCel45A is lacking a
counterpart to the aspartic acid assigned as catalytic base in
subfamily A and B, suggesting that the enzyme may utilize a
different mechanism.908 However, in common with other
inverting GH families, the assignment of the catalytic base is
less certain than of the catalytic acid within GH45. The
proposed catalytic base is not strictly conserved throughout
subfamily A and B, and so far it has been experimentally shown
to be essential for activity in only one enzyme, HiCel45A of
subfamily A.904 Further studies are clearly needed to fully
elucidate the catalytic mechanism within GH45.
10.2. Catalytic Function
Most of the over 300 entries of GH45 in the CAZy database are
known only from their sequence. Among fungi, enzyme
characterization has been reported for about 25 members,
nearly all belonging to GH45 subfamily A. Table 15 provides a
list of characterized enzymes, pH and temperature optima, and
1411
Review
substrate specificity where determined. In most cases, the
studies are limited to assays with CMC as substrate, e.g., for
demonstration of EG activity and measurements of specific
activity, pH dependence, temperature profile, and/or thermal
stability. Fewer enzymes have been analyzed in further detail in
terms of substrate specificity, product profile, enzyme kinetics,
etc. Although available data are limited, some common
properties can be distinguished. Generally, activities are
comparable or lower on amorphous cellulose (PASC) than on
CMC and much lower or negligible on crystalline cellulose (e.g.,
Avicel and filter paper). When tested, the enzymes were also
active on mixed β-1,3/1,4-glucans (e.g., barley β-glucan or
lichenan) and glucomannan, but they were essentially inactive
on xylan, xyloglucan, laminarin, or galactomannan, and release
of aglycon was negligible from chromogenic or fluorogenic
substrates such as pNPC and 4-methylumbelliferyl-β-D-cellobio-
side.332 Cleavage of glycosidic linkages other than β-1,4 has not
been demonstrated.
In an extensive study by Vlasenko et al. discussed previously,
the substrate specificities of 10 ascomycete and 1 basidiomycete
GH45 subfamily A enzymes were compared with EGs of GH
family 5, 6, 7, 9, and 12.332 In general, the GH45s showed
comparable activity to EGs of GH5, 6, and 7 on polymeric
cellulose substrates (including PASC, Avicel, pretreated corn
stover, and CMC). The most active among the tested GH45s
were the enzymes from T. terrestris, H. grisea, Chaetomium
brasiliensis, and Sordaria f imicola. Unlike other EGs, the GH45s
were essentially inactive on the hemicellulose substrates tested
in this study (xyloglucan, xylan, arabinoxylan, mannan, and
galactomannan).
In view of the structural differences discussed above, one
might expect significant differences in enzymatic properties
between the subfamilies of GH45 to follow. Apart from
subfamily A, biochemical data are scarce and have only been
reported for two members of subfamily B (TrCel45A334 and
Penicillum decumbens Cel45A921), and one in subfamily C
(PcCel45A908). Generalizing the findings from these three
enzymes, subfamily B and C members appear to exhibit lower
cellulolytic activity relative to subfamily A. Karlsson et al.
reported that TrCel45A exhibits 2−9-fold lower activity on
Avicel, PASC, and CMC relative to TrCel5A, TrCel7B, and
TrCel12A; the enzyme produces cellotetraose as major
hydrolysis product in contrast to subfamily A, where cellobiose
dominates.334 Furthermore, TrCel45A showed higher activity
on konjac glucomannan than the other EGs. Only glucose
reducing ends were formed, demonstrating that the enzyme
does not act as a mannanase on glucomannan. In the case of P.
decumbens Cel45A the activity was highest on glucomannan
followed by PASC, CMC, and β-glucan, suggesting that the
enzyme may actually be regarded as primarily a glucomannanase
rather than a cellulase.921 Activity on glucomannan was also
observed for PcCel45A of subfamily C, but the specific activity
was about 10 times higher on β-1,3/1,4-glucans (lichenan and
barley β-glucan) followed by CMC and PASC.908 No activity on
MCC or xylan was detected after 120 h of incubation.
10.3. Similarities of GH45s to Expansins and Swollenins
The general fold of the GH45 CD is shared by cell wall acting
proteins that do not exhibit hydrolytic activity but may instead
offer some disruptive effect (e.g., plant expansins and fungal
swollenins).930,931 The structural comparison of an expansin
with GH45s (Figure 73) illustrates that their β-barrels overlap
closely. The structures are also quite similar at the GH45
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 Table 15. Biochemical Characterization of Known GH45 Cellulasesa
temp
opt (°
organism expression host enzyme pH opt C) substrate for opt substrate specificity ref comments
Chrysosporium Cel45A 5 70 CMC CMC, filter paper, β-glucan, Avicel, xylan Emalfrab et al., 2003715 Ascomycota
lucknowense C1
Crinipellis scabella Cel45A PASC, Avicel, BC, pretreated corn stover, CMC, xyloglucan, Shulein et al., 2005,922 Basidiomycota; retains 25% activity at
Chemical Reviews

xylan, arabinoxylan, mannan, galactomannan Vlasenko et al., 2010332 70 °C

Humicola grisea Cel45A PASC, Avicel, BC, pretreated corn stover, CMC, xyloglucan, Vlasenko et al., 2010332 Ascomycota; retains 40% activity at
xylan, arabinoxylan, mannan, galactomannan 80 °C
Humicola grisea Aspergillus ory- Egl3 5 60 CMC CMC, Avicel Takashima et al., 2007915 ascomycota; retained >75% activity at 80
var. thermoidea zae °C for 10 min; stable at pH 3−12 at 4 °
C for 20 h
Humicola grisea Aspergillus ory- Egl4 6 75 CMC CMC, Avicel Takashima et al., 2007915 Ascomycota; Retained >75% activity at
var. thermoidea zae 80 °C for 10 min; stable at pH 3−12 at
4 °C for 20 h
Humicola insolens Cel45A CMC, PASC, Avicel, BC, pretreated corn stover, xyloglucan, Dalboege et al., 2007,923 Ascomycota; retains 83% activity at
DSM 1800 xylan, arabinoxylan, mannan, galactomannan, azurine cross- Vlasenko et al., 2010332 80 °C
linked hydroxyethylcellulose
Macrophomina Cel45A CMC, PASC, Avicel, BC, pretreated corn stover, xyloglucan, Shulein et al, 2005,922 Ascomycota; retains 22% activity at
phaseolina xylan, arabinoxylan, mannan, galactomannan Vlasenko et al., 2010332 70 °C
Melanocarpus albo- Cel45A 6−7 70 hydroxyethylcellulose CMC; hydroxyethylcellulose Miettinen-Oinonen et al., Ascomycota; retained near 50% activity
myces 2004,502 Szijártó et al., at pH 10; retained >60% activity at
2008503 70 °C
Mucor circinelloides Escherichia coli MCE1 7 45−50 CMC CMC, Avicel Baba et al., 2005906 Zygomycota
FERM P-17446
Mucor circinelloides Escherichia coli MCE2 7 45−50 CMC CMC, Avicel Baba et al., 2005906 Zygomycota

1412
FERM P-17446
921
Penicillium decum- Pichia pastoris Cel45A 5.0−3.5 60 60 konjac glucomannan konjac glucomannan, PASC, CMC-Na, barley β-glucan, xylan, Liu et al., 2010 Ascomycota; subfamily B; 90% relative
bens 114-2 and CMC Avicel activity retained at 70 °C and 30% at
80 °C
Phanerochaete Pichia pastoris Cel45A PASC, CMC, lichenan, barley β-glucan, glucomannan Igarashi et al, 2008908 Basidiomycota; subfamily C
chrysosporium K-
3
Phialophora sp. G5 Pichia pastoris EgGH45 6.0−8.0 60 CMC-Na CMC-Na, barley β-glucan, Avicel, filter paper Zhao et al., 2012723 Ascomycota
Phycomyces nitens Escherichia coli PCE1 6 50 CMC CMC Shimonaka et al., 2004924 Zygomycota
FERM P-17447
Piromyces equi Cel45A 6.5 70 CMC CMC, PASC, barley β-glucan, lichenin Eberhardt et al., 2000829 max activity at 70 °C, though dropped to
10% after 20 min; stable for at least 1 h
at 65 °C
Rhizopus oryzae RCE I 5−6 55 CMC CMC, Glc33, Glc4/Glc5/Glc6 Murashima et al. 2002925 Zygomycota
FERM BP-6889
Rhizopus oryzae RCE 2 5−6 55 CMC CMC, Glc33, Glc4/Glc5/Glc6 Murashima et al. 2002925 Zygomycota
FERM BP-6889
Rhizopus oryzae Saccharomyces RCE 3 7.7 50 CMC CMC, Avicel, Glc4/Glc5/Glc6 Moriya et al. 2003926 Zygomycota; >90% activity pH 5−7.7
FERM BP-6889 cerevisiae
Staphylotrichum STCE1 6.0 60 CMC CMC Koga et al., 2008927 Ascomycota
coccosporum IFO
31817
Syncephalastrum Pichia pastoris CBH I 5−6 70 CMC CMC, azurine cross-linked hydroxyethylcellulose Wonganu et al., 2008928 Zygomycota; retained >80% activity after
racemosum 1 h at 80 °C, and >50% for 4 h at
BCC18080 70 °C
Thielavia terrestris Cel45A CMC, PASC, Avicel, BC, arabinoxylan, mannan Shulein et al, 2005,922 Ascomycota; retains 12% activity at
Review

Vlasenko et al., 2010332 80 °C

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catalytic center. There is no obvious counterpart to the catalytic

Ascomycota; retained 70% of activity at

base of GH45 subfamily A and B, but the catalytic acid and

pH 5 and 40−70 °C; subfamily B

Ascomycota; retains 12% activity at

surrounding residues of GH45s are conserved in the
ThrXxxTyr/Phe and HisXxxAsp motifs in the β1 and β5
strands (Thr6, Tyr8, His119, and Asp121 in HiCel45A; Figure
comments

69), both in expansins and swollenins and also in other classes of

proteins recently discovered in fungi as discussed below. The
conservation of the catalytic acid and its surroundings suggests
Basidiomycota

80 °C that the machinery for protonation of a glycosidic oxygen may

play an important role in their mechanism of action.
Expansins were first discovered and characterized by
Cosgrove and co-workers as proteins responsible for cell wall
Vlasenko et al., 2010332

extension.931 In one of the original functional studies of

Karlsson et al., 2002334

Shulein et al, 2005,922

expansins, McQueen-Mason and Cosgrove demonstrated that

Schauwecker et al,

these proteins were able to mechanically weaken filter paper.931

ref

Soon after, the same authors showed that the addition of

1995929

hemicellulose greatly improved the binding of expansins to

crystalline cellulose, suggesting that the proteins might target
cellulose and hemicellulose simultaneously.932 Many studies
have followed these original reports related to the potential
CMC, PASC, Avicel, BC, pretreated corn stover, xyloglucan,

mechanisms of expansins, including structure and function

studies,933−938 hints regarding the molecular mechanism of
CMC, PASC, Avicel, Glc3/Glc4/Glc5, barley β-glucan,

expansin action in plant cell wall expansion,930,939−942 and their

ability to synergize with GHs in cellulose or biomass hydrolysis.
xylan, arabinoxylan, mannan, galactomannan

We note that observations of synergy with cellulases has been

substrate specificity

significantly mixed, with studies ranging from little to no synergy

to those suggesting substrate disruption, the latter often in the
absence of quantitative results or with only minor increases in
synergy when substrate conversion is quantified.943−947 More
glucomannan, filter paper

recently, expansins have been shown to synergize more

effectively with xylanases.948 Multiple families of expansins
have also been characterized on the basis of available sequence
data with the primary expansins studied to date termed α- and β-
expansins.308,949−951 The nomenclature of expansins has been
described by Kende et al.952 Expansins are vital to and
CMC

ubiquitous in plants. Plant genomes typically contain on the

order of 30 or more expansin genes, the majority of which are α-
All GH45 enzymes belong to subfamily A unless otherwise noted in the comments.
substrate for opt

expansins. Expansin-like proteins are also found in prokaryotes

and various nonplant eukaryotes suggesting that they are likely
important for alternate functions in nonplant organisms, such as
in root colonization.953−955
β-glucan

The protein structures of three expansins are currently

available: two group-1 pollen allergen plant β-expansins from
Phleum pratense (1N10, Major Timothy grass pollen allergen Phl
opt (°
temp

C)

P1; Fedorov, Ball, Leistler, Valenta, Almo, unpublished) and Zea

60

mays (ZmEXPB1; 2HCZ),938 and the bacterial EXLX1 from

Bacillus subtilis (3D30).937 The structures and sequence
pH opt

comparisons reveal a canonical two-domain fold of expansins.

5

An N-terminal double-psi β-barrel domain of around 110−120

residues, homologous to the GH45 catalytic module, is packed
enzyme
Cel45A

Cel45A

against a C-terminal Ig-like β-sandwich domain of ∼100

Egl1

residues; the domains are aligned to form a ∼60 Å long

putative polysaccharide-binding surface (Figure 73). Recent
expression host

structures of the bacterial EXLX1 in complex with β-1,3/1,4-

glucan and cellulose oligosaccharides show a new type of ligand-
mediated dimerization, where the oligosaccharide is sandwiched
Table 15. continued

between the Ig-like domains of two EXLX1 proteins in opposite

polarity.935 Both Yennawar et al.938 and Kerff et al.937 provide
Volutella colletotri-
Trichoderma reesei

detailed analyses and discussions of structure and sequence

Ustilago maydis
organism

similarities and differences between expansins, GH45s, and

other related proteins. Among GH45 structures, MeCel45 is
choides

most closely related to expansins,937 and GH45 subfamily B

shows higher sequence similarity with expansins than subfamily
a

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Figure 73. GH45 subfamily B is more similar to expansins than subfamily A. (A) Surface representation of the structure of HiCel45A (4ENG) in
subfamily A, with catalytic residues Asp10 and Asp121 in red and Trp18 at subsite −4 in green. (B) Surface of MeCel45A (1WC2) in subfamily B with
corresponding residues highlighted, Asp24, Asp131, and Trp64. (C) Surface of β-expansin EXPB1 from Zea mays (2HCZ), with residues Asp107 and
Trp194 highlighted. (D) Superposition of MeCel45A (1WC2; green) with Asp24, Asp132, and Trp64 shown, and expansin EXPB1 (2HCZ; purple)
with residues Asp107 and Trp194 shown. (E) Close-up of the active site of HiCel45A (blue stick), MeCel45A (green stick), and ZmEXPB1 (magenta
stick) showing the tryptophan residue at subsite −4 (Trp18/64/194), the proposed catalytic base (Asp10/24), and the catalytic acid (Asp121/132/
107) surrounded by conserved tyrosine, threonine, and histidine residues. The two cellotriose ligands in the 4ENG structure (white carbon atoms) are
included in panels D and E.

A. The catalytic center motifs ThrXxxTyr and HisXxxAsp are
conserved, but some loops are shorter in the expansins making
the putative sugar-binding surface even more shallow and nearly
flat, similar to LPMOs as will be discussed in section 11. This
may indicate that expansins act in very close proximity to an
insoluble substrate surface.
The name “swollenin” was coined by Saloheimo, Pentillä, and
co-workers at VTT, Finland, upon discovery of a new protein,
SWO1, from T. reesei that shows structure disruption effects on
cotton fibers and filter paper.309 The expression of TrSWO1 was
found to be highly upregulated under cellulase-inducing
conditions pointing to an important role in biomass
degradation. No structure of any swollenin is known to date,
but sequence similarities indicate that the C-terminal half of the
protein is homologous to expansins with one GH45-like and
one Ig-like domain. In addition, the 475-residue TrSWO1
protein contains an N-terminal family 1 CBM followed by a
typical Ser/Thr-rich linker, and a region/domain of unknown
structure and function. Several insertions between the β-strands
1414
of the GH45 domain, in particular, β1/β2 and β4/β5 (Figure
69), indicate that the putative sugar-binding surface may be
more enclosed in swollenins, or that they may form an
additional domain in analogy with EcMltA discussed below.
So far, swollenins have only been found in a limited number
of ascomycete fungi (11 species in UNIPROT). In addition to
TrSWO1,956 disruptive effects on cellulose and increased
adsorption and enhanced activity of cellulases have also been
demonstrated for swollenins from Neosartorya f umigata
(formerly Aspergillus f umigatus),957 Trichoderma asperellum,958
T. pseudokoningii,959 and Penicillium oxalicum.946 Interestingly,
Wang et al. could produce bioactive T. asperellum SWO
recombinantly in E. coli by using a cellulose-assisted refolding
and purification process.958 They also devised a method to
quantitatively measure the disruption activity of the swollenin,
by using a bacterial GH5 EG with negligible activity on
untreated crystalline cellulose. More recently the effects of
TrSWO1 on pretreated corn stover were investigated.960 The
swollenin primarily disrupted the hemicellulose fraction and
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released substantial amounts of oligomeric and monomeric
sugar. With respect to cellulose hydrolysis, synergism was small
with the CBH TrCel7A or the EG TrCel5A, whereas
pronounced synergism in terms of xylose yield was observed
with TrCel5A as well as the xylanases TrXyn10A or TrXyn11A,
the latter releasing about 3 times more xylose when combined
with TrSWO1.
A new type of GH45-related fungal protein, “loosenin” from
the basidiomycete B. adusta (BaLOOS1), was discovered and
characterized recently.961 BaLOOS1 binds strongly to both
cellulose and chitin, affects the morphology of cotton fibers, and
enhances the activity of cellulases, but did not exhibit detectable
hydrolytic activity when acting alone. Fibers of Agave tequilana
bagasse, known for high recalcitrance, were hydrolyzed 7.5 times
faster by a cellulase/xylanase cocktail after BaLOOS1 pretreat-
ment. The protein is only 109 amino acid residues in length and
consists of a sole GH45-like domain, presumably similar in
structure to that of expansins. The ThrPheTyr sequence is
present in strand β1 and the catalytic acid appears to be
conserved in strand β5, although the HisXxxAsp motif is
replaced by AspLeuAsp (Figure 69). A protein-BLAST search
returned over 200 hits from fungi with about 40% or higher
sequence identities to BaLOOS1, suggesting that loosenin
proteins are ubiquitous in fungi.
Yet another group of fungal proteins related to GH45 and
expansins is represented by EnCelA from Emericella nidulans (or
A. nidulans) in the sequence alignment in Figure 69. The CelA-
encoding gene is constitutively expressed in all developmental
cycles of the fungus, but the protein is exclusively present in the
cell walls of conidiospores.962 We note that the authors use the
name EglD, while the sequence shown in the article is identical
to that of EnCelA (Q5AVE5_EMENI) and different from that
of E. nidulans EglD (Q5BCX8; EGLD_EMENI) in the Uniprot
database. Investigations of the influence of gene expression on
morphogenesis, growth, and germination of conidia indicate
that the protein may be involved in cell wall remodeling during
spore germination. The protein was extracted from conidio-
spores and assayed in vitro for CMCase activity, but no
degradation was detected.962 The GH45-like domain is
appended by a region of ∼95 residues, similar in length as in
expansins, but it is uncertain whether it folds into a similar Ig-
like domain. Furthermore, the GH45-domain is preceded by a
region of ∼150 residues rich in Ser and Thr, which may serve as
an O-glycosylated linker of a cell wall anchor. Many members
lack this N-terminal region and may represent nonanchored
homologues. However, if they may play any significant role in
lignocellulose degradation, this appears to be unknown to date.
Several of the sequences are annotated as “cellulase” but without
reference to biochemical evidence.
Significant sequence similarity is also recognized between
GH45s and a family of lytic transglycosylases, bacterial enzymes
involved in cell wall peptidoglycan processing. EcMltA from E.
coli shows 31% and 23% sequence identity with MeCel45A and
HiCel45A, respectively. The crystal structure of EcMltA reveals
a two-domain structure where the larger A domain overlaps
closely with that of GH45s (2AE0).963 However, EcMltA has a
∼140 residue long insertion between β1 and β2 of the canonical
double-psi β-barrel topology, which folds into an additional β-
barrel B domain. A large glycan-binding groove is formed at the
interface between the two domains. Again, the catalytic center is
strikingly similar to that of GH45s. The catalytic acid-associated
motifs (ThrGlyTyr and HisPheAsp in EcMltA) are highly
conserved within lytic transglycosylase family 2, and mutation of
1415
Review
Asp308 in EcMltA resulted in complete loss of activity.963
Interestingly, though, the mechanism of EcMltA differs from
that of GH45s. The EcMltA enzyme cleaves β-1,4-glycosidic
bonds in peptidoglycan with a nonhydrolytic mechanism where
the 6-hydroxyl is joined to the anomeric carbon to form
nonreducing 1,6-anhydro-muropeptides. These lytic trans-
glycosylases thus offer an example of the utilization of the
same constellation of amino acids in a nonhydrolytic
mechanism, although analogous reaction products have not
been demonstrated in GH45s, expansins, or swollenins.
Inspired by the alternate mechanism of EcMltA, the apparent
lack of a catalytic base, and the notion of a surprisingly long
distance over the −1 subsite in HiCel45A,904 we speculate that,
at least in some cases, a mechanism might be employed that
utilizes strain in the polysaccharide concomitantly with
protonation of the glycosidic oxygen as means for glycosidic
bond cleavage. Perhaps this may be combined with a
nucleophilic attack on the anomeric carbon by a hydroxyl
group on another polysaccharide resulting in lytic trans-
glycosylation with inversion of the anomeric configuration and
without formation of new reducing ends. Such a mechanism
would make sense for expansins in particular, since the activity
would be targeted to polysaccharides under tension and would
allow for rearrangements in the polysaccharide network without
compromising the integrity of the cell wall.
Finally, we conclude that conservation of the catalytic acid
and its environment seems to be a recurring theme, suggesting
that protonation and glycosidic bond cleavage are involved also
in the GH45-related proteins. However, the molecular
mechanism for GH45 subfamily C, expansins, swollenin,
loosenin, and other related proteins remain to be elucidated.
10.4. Conclusions
GH45 cellulases are clearly industrially relevant proteins given
their inclusion in commercial detergent and conditioning
formulations. However, of all GH families of fungal cellulases,
GH45 are by far the least well characterized. Accordingly, insight
into both their structure and function is relatively limited. Here,
we generalize the findings discussed above for GH45s:
(1) GH45 CDs exhibit a six-stranded, double-psi β-barrel
fold, which is among the smallest carbohydrate active
enzyme protein folds. The small size may aid in access to
small pores in the substrate.
(2) Phylogenetic analysis suggests three different subfamily
classifications (A, B, and C) may exist and are
independent of kingdom. Currently, there is no structural
representative from subfamily C.
(3) The enzymes exhibit a wide range of cell wall
polysaccharide specificities and utilize an inverting
hydrolytic mechanism for turnover.
(4) The inverting mechanism is not well-characterized in any
GH45; however, in HiCel45A, the ligand bound structure
gives rise to the hypothesis that elongation of the
substrate in the transition state may be required for
hydrolysis.918
(5) Loop regions connecting the central β-strands contribute
to specificity and ligand binding. Conformational changes
of these loops upon binding have been observed and may
be a part of the subfamily A hydrolytic mechanism.
Subfamily B has a more open active site as a result of loop
deletions relative to subfamily A.
(6) GH45s are structurally similar to a host of other proteins
including expansins, swollenins, loosenins, and lytic
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Table 16. Reported LPMO Crystal Structuresa

source and original name in PDB ion
primary citation code resolution (Å) charge brief highlights ref
Fungal LPMO Structures
Trichoderma reesei GH61B 2VTC 1.60 Ni2+ first GH61 structure reported 337
Thielavia terrestris GH61E 3EII 2.25 Zn2+ Zn-bound GH61 structure; noted similarities with aromatics on surface to family 1 967
CBMs
Thielavia terrestris GH61E 3EJA 1.90 Mg2+ Mg-bound GH61 structure 967
Thermoascus aurantiacus 2YET 1.50 Cu2+ demonstrated copper was the active metal and observed N-terminal histidine 51
GH61 N-methylation
Thermoascus aurantiacus 3ZUD 1.25 Cu2+ Cu(NO3)2-soaked GH61 to confirm copper binding 51
GH61
Phanerochaete chrysosporium 45BQ 1.75 Cu2+ first basidiomycete GH61 structure reported 993
GH61D
Neurospora crassa LPMO-2 4EIR 1.10 Cu2+ first confirmed type 2 LPMO structure reported with activity for the C4 carbon 995
Neurospora crassa LPMO-3 4EIS 1.37 Cu2+ first confirmed type 3 LPMO structure reported with activity for the C1 and C4 995
carbons
Aspergillus oryzae LPMO 4MAH 1.55 Zn2+ first reported AA11 structure. Zn cation in the active site 1024
(AA11)
Aspergillus oryzae LPMO 4MAI 1.40 Cu2+ first reported AA11 structure; Cu(I) cation in the active site 1024
(AA11)
Nonfungal LPMO Structures
Serratia marcescens CPB21 2BEM 1.55 N/A first family 33 CBM structure reported 972
Serratia marcescens CPB21 2BEN 1.80 N/A Y54A mutant of CBP21 972
Enterococcus faecalis V583 4A02 0.95 N/A apo structure of a CBM33 enzyme 980
CBM33A
Serratia marcescens CPB21 2LHS n/a (NMR) N/A NMR structure; demonstrated that CBP21 is a rigid macromolecule 990
Bacillus amyloliquefaciens 2YOW 1.80 N/A apo structure of CBM33 enzyme 991
CBM33
Bacillus amyloliquefaciens 2YOX 1.90 Cu1+ Cu-bound structure with a Cu(I) ion; reduction was induced by X-rays 991
CBM33
1+
Bacillus amyloliquefaciens 2YOY 1.70 Cu Cu-bound structure with a Cu(I) ion; reduction was induced by ascorbate 991
CBM33
Enterococcus faecalis CBM33A 4ALC 1.49 Cu2+ first reported CBM33/LPMO from family AA9 with a +2 copper oxidation state 998
collected by a specialized diffraction method
Enterococcus faecalis CBM33A 4ALT 1.49 Cu2+ photoreduced CBM33/LPMO from family AA9 with a +1 copper oxidation state 998
Streptomyces coelicolor 4OY6 1.29 Cu2+ one of two first cellulose active bacterial (AA10) LPMO structures solved 991
LPMO10B
Streptomyces coelicolor 4OY7 1.50 Cu1+ second of two first cellulose active bacterial (AA10) LPMO structures solved 991
LPMO10C
a
Both fungal and non-fungal LPMOs are included.

transglycoyslases, many of which have conserved major
components of the GH45 active site.
Much remains to be accomplished toward understanding
GH45 enzymes and their hydrolytic mechanisms. The current
dearth of information cannot even definitively address the
question of consistent mechanisms across subfamilies. Even the
simplest of questions such as the identity of the catalytic base is
likely to remain unaddressed for some time given the difficulty
in attribution this role in inverting enzymes (see GH6
discussion). Nevertheless, these enzymes and the GH45-like
counterparts play a role in both cell wall deconstruction and
generation, suggesting further examination of this interesting
family is warranted both for their potential in industrial
applications and for understanding their role in natural
biomass-degrading contexts.
11. LYTIC POLYSACCHARIDE MONOOXYGENASES
The structures of ligand-bound GHs described above marked a
turning point in our collective understanding of plant cell wall
degradation starting in the 1990s that has enabled more than
two decades of intense mechanistic research and development.
However, as discussed above, cellulose and similar polysacchar-
ides pack tightly into dense crystalline lattices on which GHs
must act. Given the inherent recalcitrance of cellulose,
1416
decrystallization of individual chains requires thermodynamic
work.130−132,580,964 Over 60 years ago, Reese and co-workers
speculated that nature likely employs additional mechanisms to
disrupt the crystalline lattice of cellulose.291 To this end, a
discovery was made in 2010 that verified this hypothesis and
overturned the conventional cellulose depolymerization para-
digm.48 Namely, the characterization of a new family of biomass-
degrading enzymes, now referred to as LPMOs,42,50,965 revealed
a completely new mechanistic approach for cleaving glycosidic
bonds in cellulose (and chitin). It was shown that LPMOs can
be quite synergistic with the traditional GH enzyme cocktails
described above, even before the basis for their mechanisms was
reported.966,967 These enzymes were previously characterized
either as family 33 CBMs or family 61 glycoside hydrolase
(GH61) cellulases from nonfungal and fungal origin,
respectively. LPMOs are now understood to be ubiquitous in
nature, to be highly upregulated during biomass degradation,
and to exhibit significant diversity both within and between
biomass-degrading organisms.60,965,968,969
Given that LPMOs are emerging as key components of
cellulase cocktails, likely due to their ability to create chain
breaks in crystalline regions of cellulose where EGs would not
be able to productively bind to the substrate, here we briefly
review the discoveries thus far in this new field. We note that in
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Figure 74. S. marcescens CBP21 structure and initial mechanistic discoveries. (A) The overall structure of S. marcescens CBP21 with the active site
residues highlighted in stick format.972 PDB code: 2BEM, chain C. His28, Glu60, Ala112, His114, and Phe187 are shown in stick format. The
coordination distances to the ion in the structure to the histidine residues are highlighted. (B) The S. marcescens CBP21 active site.972
this section we describe research into both fungal and nonfungal
LPMOs as the mechanistic developments to date are closely
linked. Unlike previous sections, we primarily follow a
chronological description of the scientific developments and
discoveries, as this field of cellulase research is quite nascent. At
the end of the section, we then summarize the current state of
the knowledge and several interesting future directions. Despite
significant progress, there is still much to learn about the
mechanisms employed by these fascinating, newly discovered
enzymes.
Lastly, the nomenclature related to these enzymes has evolved
considerably and likely will continue to do so for some
time.42,50,152 Although all enzymes described in this section are
LPMOs, we generally refer to the enzymes as was done
historically (e.g., we refer to them as “GH61” or “CBM33”,
described below, when discussing studies before the term
LPMO was coined). We note that the term LPMO is now
reasonably well accepted and conveys the salient details of their
currently reported function. Remarks on the likely nomencla-
ture for these enzymes is provided at the end of this section.
Additionaly, descriptions of structures discussed in this section
are provided in Table 16.
11.1. Initial Discoveries of Oxidative Function
Prior to 2005, family 33 CBMs from insect viruses had
previously been shown to bind to chitin, but no mechanistic
details were revealed.970,971 In 2005, Vaaje-Kolstad et al.
reported the first family 33 CBM structure from the chitinolytic
bacterium Serratia marcescens (Table 16).972 The protein was
termed “chitin-binding protein 21” (CBP21), where the “21”
denotes the fact that the protein is 21 kDa.966,972 The overall 3-
D structure of CBP21 was characterized as a “budded”
fibronectin type III fold (Figure 74). In the same study,
mutations of residues on the putative chitin-binding surface
were conducted, all of which were shown to reduce the binding
affinity to the substrate.972 The CBP21 structure harbors a metal
ion between the conserved N-terminal histidine (His28) and
another conserved histidine (His114), but its function was not
initially realized (Figure 74). Shortly after, the same group
published a study wherein they systematically demonstrated that
CBP21 dramatically boosts β-chitin conversion in the presence
of any of the three native GH18 chitinases from S. marcescens
(two chitobiohydrolases, one endo-chitinase) or in mixtures
with all three GH18 chitinases present.966 One of the conserved
1417
Review
histidine residues (His114) of CBP21 was mutated, which
resulted in complete elimination of the synergistic benefits of
action.966 A later report from Moser et al.973 additionally
demonstrated that two CBM33s from T. f usca (denoted E7 and
E8) were also able to synergize with S. marcescens chitinases in
the depolymerization of β-chitin and to a lesser extent with T.
f usca cellulases at low concentrations on filter paper assays.973
These reports marked the first in-depth biochemical and
structural studies of CBM33 proteins and, especially in the
case of S. marcescens CBP21, hinted at an unknown, powerful
mechanism for disrupting crystalline polysaccharide sub-
strates.966,972,974
In 2010, Harris and co-workers reported an in-depth study of
GH61 enzymes from the fungi T. aurantiacus and T. terrestris.967
Before the study from Harris et al., GH61s were thought to
exhibit weak EG activity, for example on CMC,487,975−977 and
their considerable synergy with the T. reesei secretome had only
been briefly reported.978 Harris and co-workers demonstrated
that either GH61 enzyme added to a native T. reesei cellulase
cocktail at low concentrations (4 mg GH61/g cellulose)
significantly enhanced the T. reesei enzyme cocktail performance
on pretreated corn stover (Figure 75).967 Indeed, the authors
report a reduction in overall protein loading was possible by a
factor of 2 if a GH61 enzyme was added, which is a reduction in
loading with significant cost savings implications for enzymatic
hydrolysis of pretreated biomass.21−23,967 Intriguingly, the
authors also showed that there was no synergy on various
clean cellulose substrates (e.g., Avicel), which at the time was
hypothesized as a mechanism of GH61 action on either
hemicellulose or lignin. The T. terrestris GH61E enzyme
structure was solved (Figure 76), which was the second
representative structure from this family (the first structural
member was T. reesei GH61B (TrGH61B) reported two years
earlier337). In both the structural reports from Harris et al.967
and from Karkehabadi et al.,337 it was speculated that GH61
enzymes are not GHs due to the lack of conserved, carboxylate
pairs in sufficient proximity required for GH action.169,177
Karkehabadi et al. also noted a structural similarity to S.
marcescens CBP21.337 In the two first GH61 structures solved,
similar to CBM33s,972 both exhibit a metal-binding site
coordinated by two histidine residues on the flat face of the
protein. Harris et al. both varied and removed the metal
altogether with EDTA, and demonstrated that the presence of a
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Figure 75. Comparison of cellulase activity after 75 h of the specified T.
reesei cellulases alone (light gray) or with T. aurantiacus GH61A
included at 10% of the total protein loading of 2.5 mg/g of cellulose
(dark gray). The T. reesei mixtures tested comprised ratios of 4.5:2.5:1.0
for Cel7A:Cel6A:Cel7B.967
divalent metal cation is essential for the observed performance
enhancements. Mutation of both of the conserved, ligating
histidine residues to alanine completely abolished activity as
measured by synergistic performance with a T. reesei cocktail on
pretreated corn stover.967 Lastly, Harris et al. noted a similarity
in the orientation and spacing of three tyrosine residues on the
flat face of T. terrestris GH61E to that of family 1 CBMs, perhaps
suggesting the putative GH61 binding face to the cellulose
surface (Figure 76).346,351,352,355,362,967 Taken together, these
studies showed that GH61s and CBM33s share similar
structural functionalities and that the metal coordination was
important for synergism with GHs.337,966,967,972
The major breakthrough in understanding the mechanistic
basis of these enzymes was reported in 2010 when Vaaje-
Kolstad et al. observed that CBP21 can depolymerize β-chitin
alone in the presence of a reducing agent and molecular oxygen
pointing toward an oxidative mechanism for CBP21.48
Specifically, oligomers were released from β-chitin that exhibited
an aldonic acid functionality at the C1 carbon. Using mass
Figure 76. T. terrestris GH61 enzyme and active site.967 PDB code: 3EII (A) An overall structural view is shown with active site residues highlighted in
yellow stick. The aromatic residues forming the putative cellulose binding face of the enzyme are shown in green stick. (B) The active site
representation illustrates the coordination distances of the zinc ion (gray sphere) to His1, His68, and two water molecules shown in red sphere.
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spectrometry experiments with isotopically labeled oxygen
atoms in either 18O2 or with H218O, the authors demonstrated
that the aldonic acid functionality incorporated one oxygen
atom from O2 and one from H2O (Scheme 3). This led the
Scheme 3. Overall Reaction for S. marcescens CBP21 Action
on Chitina
a
Separate mass spectrometry experiments with 18O-labeled molecular
oxygen and water demonstrated that S. marcescens CBP21 action
involves a tandem oxidation−hydrolysis mechanism.48
authors to postulate a mechanism wherein both oxidation and
hydrolysis occur as a result of CBP21 action. Another
noteworthy observation from this work is that cyanide
significantly inhibits CBP21 action. As cyanide is a well-
known inhibitor of O2 binding to metal centers in enzymes and
catalysts, this suggests that molecular oxygen binds directly to
the histidine-coordinated metal center in the enzyme. Addi-
tionally, superoxide dismutase does not inhibit the reaction
significantly implying that the metal center in the oxygen-bound
form of the enzyme is shielded from bulk solvent. Overall, these
results suggest that CBP21 acts directly on the surface of β-
chitin, to which it is known to bind with high affinity,966,972 and
that molecular oxygen binds to the metal center primarily when
the enzyme is actively engaged on the substrate for oxidation.48
It was noted that, given the similarity in structure and metal-
binding site between CBM33s and GH61s, these enzymes may
employ similar chemical mechanisms.48 This seminal study
marked a turning point in biomass conversion research in that
the conventional, nearly universal hydrolytic paradigm was
overturned; a drastically different mechanism in the cleavage of
glycosidic bonds in polysaccharides was revealed.48 It is now
generally hypothesized that these oxidative enzymes produce
chain breaks in crystalline regions of cellulose (and chitin),
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Figure 77. T. aurantiacus GH61 structure. (A) Overall structure of the T. aurantiacus GH61 (LPMO) with the active site residues shown in stick
format.51 PDB code: 2YET, chain A. His1, His86, Gln173, Tyr175 shown in stick format. (B) Active site of the T. aurantiacus GH61 (LPMO)
enzyme.51 Note that the Nε2 atom of the N-terminal histidine exhibits a methyl group of unknown function. The two red spheres coordinating the
copper ion are water molecules. The coordination geometry of the copper ion in this enzyme is octahedral, indicating a +2 oxidation state.
which likely is synergistic with conventional GH enzymes due to
the presence of additional attachment and detachment sites for
processive GHs. EGs, on the other hand, are thought to act
primarily in amorphous regions where the substrate is more
accessible for complexation.557
From the initial discovery of oxidative activity in CBP21,
many studies have been subsequently published to characterize
various features of the LPMO catalytic mechanisms, to
understand substrate specificity and regioselectivity, and to
understand the range of reducing agents able to potentiate this
oxidative activity. Soon after the initial characterization of
CBP21 oxidative action, Forsberg et al. reported that a two-
domain CBM33 enzyme from Streptomyces coelicolor A3(2) is
active on cellulose.979 Similar to CBP21, the S. coelicolor CBM33
primarily produced oxidized oligomers that had an even number
of glycans in the product. Additional chitinolytic CBM33
structures have been reported from the same group, which
exhibit a similar product profile as the one of S. marcescens
CPB21.980
11.2. Mechanistic and Structural Studies
Subsequently, four studies were published in rapid succession in
2011, several of which demonstrated that GH61s employ copper
as the active metal and demonstrated that multiple chemical or
enzymatic reducing agents can promote oxidative activ-
ity.49−51,981 Langston et al. demonstrated that CDH is able to
act as a reducing agent for GH61 activity (specifically for the T.
aurantiacus GH61 enzyme and H. insolens CDH).49 It has long
been known that CDHs are coregulated with cellulases, and
their specific function beyond cellobiose oxidation has been the
subject of intense study.982,983 CDH consists of a heme domain
and a flavin adenine dinucleotide (FAD) domain. The FAD
domain of CDH is responsible for a 2-electron oxidation of
cellobiose, and the heme domain is believed to transfer the
electrons from the FAD domain to another electron acceptor,
including in this case to GH61 enzymes. Structures of the heme
and FAD domains are known,984,985 and kinetic measurements
of electron transfer rates have been reported.986,987 Langston et
al. also showed that the natively expressed CDH and GH61
enzymes from T. terrestris are synergistic in cellulose
depolymerization.
With the GH61 from T. aurantiacus expressed in A. oryzae,
Quinlan et al. employed isothermal titration calorimetry,
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electron paramagnetic resonance (EPR) spectroscopy, crystallo-
graphic studies, and activity assays to demonstrate that copper is
the active metal.51 They found that copper binds very strongly
to the metal-binding site (the Kd was shown to be tighter than 1
nM),51 and they also observed a methylation at the Nε2 atom of
the N-terminal histidine residue in the enzyme active site
(Figure 77). This histidine N-methylation was also present in
the previously reported T. terrestris and T. reesei GH61
structures,337,967 but was not described in the papers describing
the structures. In unrelated systems, this post-translational
modification has been shown to modify copper-binding
systems,988 perhaps by modulating the binding affinity of
copper via a pKa shift of the histidine residues989,990 or by
modifying the overall electronic structure of the system such
that the oxygen activation and reactivity is altered.989−991
Overall, the active site geometry in the T. aurantiacus GH61
structure appears to harbor a Cu(II) cation and displays classic
Jahn−Teller distortion with elongated coordination bonds in
the axial positions. In the equatorial plane, the copper ion is
ligated in a bidentate fashion to the N-terminal histidine and
monodentate to a second histidine residue and a polyethylene
glycol hydroxyl group, the latter from the crystallization buffer.
The authors coined the term “histidine brace” for this structural
motif that binds copper.51 Via activity assays, they also
demonstrate that copper is essential to the reactivity of the
enzyme, and that reducing agents such as ascorbate and gallate
can potentiate activity.51 Action at the C6-carbon was also
postulated, but evidence of this type of activity was not reported
and has not been definitively verified to date in the open
literature. Although at least one other study has presented
indirect evidence that C6-oxidation may occur, it may be that
these observations are actually C4 oxidation, as discussed
below.992
Shortly after, Phillips et al. reported the detailed character-
ization of a N. crassa GH61 enzyme and its interaction with
CDH from the same organism.50 Specifically, they knocked out
the CDH-expressing gene in N. crassa, which resulted in a
significant reduction in the cellulolytic performance of the
secreted enzyme cocktail in the mutant strain. Add-back studies
with CDH from M. thermophila resulted in a full rescue of
performance suggesting that CDH is a primary driver of biomass
depolymerization in N. crassa.50 The authors then systematically
demonstrated that both metals and oxygen were important for
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Scheme 4. Originally Proposed Catalytic Mechanism for LPMO Action50,312,a
a
The final hydroxylated product (top left) will undergo a rapid elimination reaction to form a C1-lactone and cleave the glycosidic bond. It is noted
that an oxidative attack at the C4 carbon follows the same overall mechanism in terms of the elementary steps and differs only in the position of the
oxidative attack and the final product (namely a 4-keto sugar instead of a C1-lactone).
the CDH enhancement of the N. crassa secretome by loading
with EDTA and running the hydrolysis reaction anaerobically,
respectively. Given these observations, the authors then
analyzed GH61 proteins in the N. crassa secretome with tryptic
digestions, LC-MS/MS analysis, and inductively coupled
plasma-atomic emission spectroscopy (ICP-AES), which
demonstrated that the GH61 enzymes from N. crassa bind
copper in a 1:1 ratio. On the basis of the detailed chemical
analysis conducted in their study, Phillips et al. thus
independently concluded that copper was the active metal in
GH61 enzymes and that CDH potentiates its activity. By
analyzing multiple GH61s, the authors also discovered the
presence of 2 types of specificities: some N. crassa GH61s
produced C1-oxidized species (aldonic acids), dubbed “type 1”,
whereas some GH61s were suggested to form 4-keto sugars via
oxidation at the C4 carbon (dubbed “type 2”). Phillips and
colleagues proposed that GH61 and CBM33 enzymes should be
termed “polysaccharide monooxygenases”, which was later
modified with the term “lytic”,42 thus generally becoming
referred to as LPMOs.965 This study was also the first to propose
a chemical mechanism for GH61 action, illustrated in Scheme 4.
The mechanism proposed by Phillips et al. consists of the
following steps: reduced CDH transfers an electron to the
“resting” Cu(II)-bound LPMO to form Cu(I), which then binds
molecular oxygen (Scheme 4). Oxygen then abstracts an
electron from Cu(I) to form a Cu(II)−superoxo species
(Cu(II)−O−O•), which can then abstract a hydrogen atom
from either the C1 or C4 carbon of the substrate to form a
Cu(II)−hydrosuperoxo species (Cu(II)−O−OH) and a radical
on the substrate centered at the point of hydrogen atom
abstraction. Another electron and proton are transferred from
CDH to form water and a Cu(II)−oxyl species (Cu(II)−O•).
The Cu(II)−oxyl species then hydroxylates the substrate, which
undergoes a spontaneous and rapid elimination reaction to form
a lactone or 4-keto sugar at the C1 or C4 carbon, respectively,
resulting in glycosidic bond cleavage.50 The lactone form can
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then further, either spontaneously or via enzymatic catalysis,
form an aldonic acid, which aligns with the mass spectrometry
experiments reported by Vaaje-Kolstad et al. in the initial report
of CBP21 employing an oxidative catalytic mechanism.48
Directly following the reports from Quinlan et al.,51 and
Phillips et al.,50 Westereng and co-workers reported that P.
chrysosporium GH61D, expressed heterologously in Pichia
pastoris, is also a copper-dependent enzyme that is primarily
specific to glycosidic bond cleavage at the C1 carbon in
cellulose.981 Likely due to the expression in P. pastoris, the N-
terminal histidine did not exhibit N-methylation as observed
earlier in the T. aurantiacus,51 T. terrestris,967 or T. reesei
structures.337 However, even without the N-methylation, the P.
chrysosporium GH61D was still active on cellulose.981 The same
observation has also been made for two GH61s from Podospora
anserina, also expressed in P. pastoris.992 Direct comparisons of
an oxidative enzyme with and without this post-translational
modification to date have not been reported in the open
literature. A more recent follow-up study was also published
wherein the crystal structure of P. chrysosporium GH61D
produced from P. pastoris was reported.993 This LPMO structure
was the first reported from a basidiomycete fungus, which seems
to utilize LPMOs quite ubiquitously in wood degradation.994
These initial mechanistic and structural characterizations of
LPMO enzymes in 2011 demonstrated unequivocally that
copper is a highly active cofactor in LPMO action, that CBM33s
and GH61s share some commonality in their metal-binding sites
that suggests an overall similar mechanism, and that a reducing
agent, either enzymatic or from small molecules, and molecular
oxygen are needed for catalysis by LPMOs.48,50,51,979,981 In
2012, more reports began to generally refer to these enzymes as
“LPMOs”, and several additional details regarding LPMO action
were reported. Beeson et al. reported an additional character-
ization of N. crassa LPMO action wherein they more definitively
assigned the type 2 LPMO products to be 4-keto sugars,312 as
suggested earlier in Phillips et al.50 Soon after, the same group
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published two structures of natively expressed N. crassa LPMOs
(GH61s), one of which (a type 2 LPMO) was shown to be
specific for oxidation at the C4 carbon, and the other of which is
a type 3 LPMO that carried out oxidation both at the C1 and C4
carbons in cellulose.995 In both structures an N-methylation of
the N-terminal histidine reported earlier was observed.51,995
Interestingly, the active site in both enzymes displays an oblong
molecule coordinated end-on (η1) to the copper cation. The
Cu−O1 distances reported are 2.96, 2.92, and 3.44 Å (chains A
and B in N. crassa LPMO-2 and chain A in N. crassa LPMO-3,
respectively).995 For the species in N. crassa LPMO-2, assuming
that the species is an O−O species, the O−O bond distance is
1.16 Å. This observation led the authors to speculate that this
may be a dioxygen species, namely superoxo, weakly bound to
the copper atom.995 However, this observation has been
questioned given the distance from the copper ion to the
putative O1 oxygen. In Cu(II)−O−O• superoxide species, the
Cu−O bond distance should be around 1.33 Å.996 Nonetheless,
another interesting observation was made in the N. crassa
LPMO-3 structure; the molecule crystallizes as a face-to-face
dimer, and a tyrosine residue near the active site of its
neighboring LPMO was hydroxylated to 3,4-dihydroxyphenyla-
lanine, perhaps suggesting that the X-rays reduced copper and
activated the enzyme, resulting in hydroxylation of the nearby
tyrosine residue in the neighboring molecule.995
The study from Li et al. also presented sequence alignments
grouped by LPMO type to ascertain which residues are
important for regioselectivity.995 On the basis of these
alignments, the authors described potential enzyme−substrate
interactions that give rise to the difference LPMO types via
docking the LPMOs onto the cellulose surface. They noted
several differences between type 1, 2, and 3 LPMOs including
differences in the number and placement of aromatic residues
and putative N-glycans near the binding surface, informed by
previous studies on family 1 CBM−cellulose interac-
tions.346,352,371 More recently, the same group has developed
further insights into the regioselectivity of LPMOs. Vu et al.
combined phylogenetic analysis over a much larger set of
LPMOs with experimental studies of additional N. crassa
LPMOs to identify structural motifs that are important for
regioselectivity.997 Vu et al. demonstrated that type 3 LPMOs
exhibit an extra loop near the N-terminus, consisting of
approximately 12 amino acid residues. Removal of this loop in
an N. crassa LPMO resulted in the production of primarily
aldonic acids (i.e., oxidation at the C1 carbon or conversion to a
type 1 LPMO), suggesting that this loop is important for C4
specificity.997 Multiple additional conserved residues were found
from their phylogenetic analysis, all of which now highlight the
need for a large mutation campaign to further understand the
structural aspects for LPMO regioselectivity. Interestingly, the
comprehensive sequence analysis of fungal LPMOs from Vu et
al. highlights two subfamilies of LPMOs that they mention have
no structural or functional characterization to date (Figure 4 in
ref 997), which is perhaps an area ripe for discovery of new
LPMO activities or substrate specificities.997
In many of the earlier LPMO studies, it was assumed that the
flat face of LPMOs is the primary binding face to crystalline
substrates. To verify this hypothesis and to gain further insights
into the copper binding in bacterial LPMOs (CBM33s),
Aachmann and co-workers conducted a detailed NMR and
isothermal titration calorimetry study on S. marcescens
CBP21.990 Interestingly, they applied an elegant NMR method
to demonstrate that the flat face harboring the copper-binding
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site is indeed the binding face to crystalline chitin. Additionally,
it is likely that LPMOs first require in situ reduction of Cu(II) in
the active site to “activate” the enzyme for oxygen binding, as
also proposed by Phillips et al.50 To understand the binding
affinity differences between monovalent and divalent copper,
Aachmann et al. used competition assays to demonstrate that
Cu(I) binds more strongly to CBP21 than Cu(II) (1.2 nM
compared to 55 nM, respectively). Similar to their previous
report,48 the authors also show that cyanide directly inhibits
CBP21, but with NMR, they demonstrate that cyanide interacts
with the copper ion, thus more definitively showing that the
metal takes place directly in the reaction.990
To further elucidate the nature of LPMOs with Cu(I) bound
to the active site, Hemsworth et al. recently reported a structure
photoreduced with X-rays.991 Similar to the study from
Aachmann et al.,990 they demonstrate Cu(II) binds with very
high affinity to a CBM33 from Bacillus amyloliquefaciens, ranging
between 6 nM at pH 5 and 80 nM at pH 7.991 Interestingly, they
also measure the melting temperature of the enzyme with and
without copper bound, which shows an astounding 20 °C
increase in the presence of copper. An X-ray photoreduced
structure exhibits a T-shaped configuration, indicative of a
Cu(I)-bound state. A primary outcome from the work of
Hemsworth et al. that should be noted in structural studies of
LPMOs is that X-rays can reduce copper, and thus proper care
should be taken when analyzing the active site geometries to
note the coordination of the copper atom, as proper analysis of
the coordination geometry can immediately suggest the
oxidation state of the copper ion.991 Lastly, the authors give
significant mechanistic weight to the differences in nonfungal
LPMOs (CBM33s) and fungal GH61s in this study, as discussed
in more detail below.991
As a further illustration of the sensitivity of LPMO active sites
to X-ray radiation, Gudmunsson et al. also recently reported a
series of bacterial LPMO structures from Enterococcus faecalis
CBM33A (EfaCBM33A) along the reduction pathway from
Cu2+ to Cu1+.998 The original EfaCBM33A structure was solved
previously in an apo form.980 A specialized photoreduction
method using helical X-ray diffraction was employed to collect
structures along an increasingly intense dosage profile. These
structures definitively revealed the change in LPMO active site
architecture in the two oxidation states, namely with the Cu2+
state exhibiting a trigonal bipyramidal geometry and the Cu1+
state in a T-shaped configuration. Comparison of these
structures to similar small-molecule crystal structures bound
to copper unequivocally demonstrated that the oxidation states
of copper in the low and high dosage structures are Cu2+ and
Cu1+. Moreover, the method employed in this study provides a
straightforward means to solve LPMO crystal structures in both
copper oxidation states.
Until the end of 2013, all LPMOs reported in the literature
were only active on the insoluble substrates cellulose and chitin.
Although likely an important feature of LPMOs utilized by
biomass-degrading organisms, this substrate preference creates
significant challenges in the evaluation of kinetics, study of the
catalytic mechanism, and detailed molecular-level character-
ization of enzyme−substrate interactions. Isaksen et al. recently
described the biochemical characterization of the first reported
LPMO acting on soluble oligosaccharides of cellulose.999
Namely, an N. crassa LPMO (NCU02916) or NcrLPMO9C
was found to exhibit substantial activity on cellopentaose and
cellohexaose with specificity for the C4-carbon. For cellopen-
taose oxidation, a nonoxidized trimer and a C4-oxidized dimer
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Scheme 5. Overall Reaction Scheme for C1 and C4 Oxidation of Cellulose by LPMOs and Subsequent Hydrolysisa

a
(A) C1 oxidation produces a lactone species, and subsequent hydrolysis produces an aldonic acid functionality at C1 overall resulting in glycosidic
bond cleavage.48,50,51 (B) C4 oxidation produces a 4-keto sugar which can then undergo hydrolysis to form a gemdiol species.50,312,999

Scheme 6. Copper−Oxyl Catalytic Mechanism Examined by Density Functional Theory Calculationsa

a
Density functional theory calculations predicted that a Cu(II)-oxyl species is the most likely reactive oxygen species (ROS) in the LPMO catalytic
mechanism, which has been predicted previously to exhibit significant oxidative power. The barrier for this reaction mechanism is predicted to be
18.8 kcal/mol. The rate-limiting step is hydrogen abstraction from the substrate. H-abstraction from the C1 and C4 carbon are predicted to be
energetically equivalent.1003

are observed, suggesting binding in a −3 to +2 mode in the
enzyme using the nomenclature for carbohydrate binding
sites.175,176,999 This enabled more detailed characterization of
the reaction productions with NMR spectroscopy than was
possible before, which when combined with MS/MS analysis
definitively reveals a 4-keto sugar product that further
hydrolyzes to a geminal diol in solution (Scheme 5).999 Even
more importantly, this discovery enables incredible potential for
experimental elucidation of the LPMO catalytic mechanism. As
a result, substrate-bound structures may now be possible via
NMR or X-ray crystallography. The development of LPMO
kinetic assays will likely also follow soon, which will enable
experimental determination of the rate-limiting steps in LPMO
action. By using unreactive analogues (e.g., cyanide in place of
oxygen), isolation of reactive intermediates will likely be
1422
possible, which will enable dramatic advances in our collective
understanding of these important oxidative enzymes. Using the
same enzyme, the Eijsink group subsequently reported that it is
active on xyloglucan.1000 This report highlights the potential
scope of LPMO substrate specificity, and likely suggests that
oxidative depolymerization of polysaccharides is likely more
general than for just cellulose and chitin. Indeed, a recent study
has identified an LPMO that is active on starch.1001
As noted in multiple studies since the initial report that
LPMOs were oxidative enzymes, understanding the catalytic
mechanism is one of the most important outstanding problems
for LPMOs.48,50,51,312,991,995,1002 To that end, the first report of
the LPMO catalytic mechanism was recently published.1003
Using density functional theory calculations, Kim et al. used an
active site, or “theozyme” model,1004 of the T. aurantiacus
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Figure 78. A. orzyae AA11 LPMO structure. (A) Overall structure of the A. orzyae LPMO structure with the active site residues shown in stick
format.1024 PDB code: 4MAI. His1, Ala58, His60, Glu138, Tyr140 shown in stick format. (B) Active site of the A. orzyae LPMO enzyme. The copper
ion coordination state indicates that the copper ion is in a +1 oxidation state.
LPMO51 to first examine how oxygen binds to a Cu(I)-bound
state in the LPMO active site. With this activated structure, two
catalytic mechanisms for hydrogen abstraction and substrate
hydroxylation were compared with transition state calculations,
one with a copper−superoxo reactive oxygen species (ROS) and
the other utilizing a copper−oxyl ROS. For oxygen binding to
the LPMO active site, it was predicted that oxygen binds end-on
(η1) to copper with an O−O bond distance of 1.31 Å, which is in
good agreement with the ideal bond length of Cu(II) superoxide
species of 1.33 Å.996 From there, a “superoxo” mechanism was
tested wherein the LPMO−Cu(II)−superoxo species abstracts a
hydrogen atom from C1 (or C4) carbon of a cellobiose unit,
which was chosen to represent the cellulose chain. This reaction
forms a radical centered on the substrate carbon atom (C1 or
C4) and an LPMO−Cu(II)−hydroperoxo species. The O−O
bond then breaks, and the hydroxyl group undergoes a rebound
mechanism to form a covalent bond with the C1 (or C4)
carbon. This results in a hydroxylated substrate and an LPMO−
Cu(II)−oxyl species, and the oxyl group is then reduced to
water by action of a reducing agent. The potential energy surface
(PES) for this reaction exhibits a barrier of 43.0 kcal/mol,
regardless of attack at either the C1 or C4 carbon.1003 Given
such a high barrier for a Cu(II)−superoxo mechanism, it was
hypothesized that a much more powerful ROS was needed for
cellulose hydroxylation. Cu(II)−oxyl species have previously
been predicted to exhibit a much stronger oxidative character
than Cu(II)−superoxo.1005,1006 Given the likely very short
lifetime of Cu(II)−oxyl, it has only been experimentally isolated
in a single study to date.1007 Nonetheless, this species has
previously been implicated in methane and aromatic hydrox-
ylation reactions.1008−1010 Thus, Cu(II)−oxyl was subsequently
hypothesized to be the ROS in LPMO action. The reaction
cycle was proposed as follows (Scheme 6): a reducing agent first
acts on the LPMO−Cu(II)−superoxo species to produce water
and an LPMO−Cu(II)−oxyl species. LPMO−Cu(II)−oxyl
abstracts a hydrogen from the substrate to form a LPMO−
Cu(II)−hydroxyl species, which then undergoes an oxygen-
rebound mechanism to hydroxylate the substrate. The overall
barrier for this reaction mechanism was calculated to be 18.8
kcal/mol, which is a much more feasible energy barrier at
biological conditions than 43.0 kcal/mol for a “superoxo”
mechanism. The rate-limiting step in the Cu(II)−oxyl
mechanism is C−H abstraction from the substrate, and
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abstractions from the C1 and C4 carbon are energetically
quite similar.1003 Although the two proposed reaction
mechanisms from Kim et al.1003 draw heavily on the proposed
catalytic cycle from Phillips and colleagues50 (Scheme 4) and
the study of other copper oxygenases (refs 1005, 1006, 1009,
1011−1023), there are some important contrasting features.
Namely, the mechanisms proposed by Kim et al. do not require
formation of two radicals simultaneously, and do not employ a
reducing agent at disparate parts of the catalytic cycle.1003
Additionally, N-methylation of the N-terminal histidine was also
studied with the theozyme approach, but no appreciable
differences were predicted on the basis of the theozyme
method. This study provided the first detailed examination of
the LPMO catalytic mechanism, and implicated a ROS for
LPMO action of substantial oxidative power,1005,1006 concom-
itant with the incredibly strong glycosidic bonds in poly-
saccharides.71 Certainly, however, more work remains to be
conducted to fully characterize the LPMO catalytic mechanism.
Given the reclassification of GH61s and CBM33s as LPMOs,
the curators of the CAZy database recently published an update
in which many oxidative biomass-degrading enzymes including
LPMOs were catalogued.152 With the diverse activity of this
portfolio of enzymes, the large suite of oxidative enzymes was
given the generic title “auxiliary activity” or AA. Fungal LPMOs,
formerly GH61s, are classified as AA9 enzymes whereas
nonfungal LPMOs are classified as AA10 enzymes.152
Additional families of LPMOs have also recently begun to
emerge. Hemsworth and colleagues reported the discovery of a
new LPMO family, classified as AA11 on the CAZy database.1024
The authors indicate that this class of LPMOs is phylogeneti-
cally distinct from the currently classified oxidative enzymes, and
characterize a specific member of this new family, which is a
chitinolytic LPMO from A. oryzae that forms aldonic acids. The
structure of the AA11 LPMO is overall quite similar to both
previously characterized fungal and nonfungal LPMOs. A
conserved alanine residue is contained in the active site, similar
to nonfungal LPMOs, and a tyrosine residue resides axial to the
copper ion. On the basis of this observation and detailed analysis
of the EPR spectra, the authors suggest that AA11 LPMOs are
intermediate between the two known families (Figure 78).
In 2014, a report from the Eijsink group was also published
which demonstrated that AA10 LPMOs also exhibit a broader
regioselectivity than just C1 oxidation.991 In a structural and
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biochemical study from Forsberg et al., the authors solved high-
resolution structures of a pair of AA10 LPMOs from S. coelicolor,
and showed that one was a C1 oxidizer and the other specific to
C4 oxidation. The enzymes were also shown to be synergistic in
their degradation of cellulose. They also demonstrated that the
copper affinities, redox potentials, and EPR spectra are quite
similar for both the C1 and C4 oxidizing LPMOs. The outcome
from this study based on a structural comparison of the active
sites is that regioselectivity is likely directed by residues beyond
the first “shell” from the copper binding site of LPMOs, similar
to hypotheses from AA9 LPMOs.
From a more industrial standpoint, additional discoveries
have been made related to LPMO action. Related to the original
observation that LPMOs do not synergize with GH cocktails on
clean cellulose substrates from Harris et al.,967 Dimarogona and
colleagues demonstrated that the presence of lignin specifically
boosts the activity of a fungal LPMO.1025 Cannella et al.
conducted a study with the LPMO-containing Cellic CTec2
cocktail (Novozymes, Inc.) at high solids loadings, and
demonstrated that up to 4% of the glucose released was
gluconic acid.626 The authors went on to further demonstrate
that the β-glucosidase in Cellic CTec2 cleaves the glycosidic
bond in cellobionic acid 10 times slower that in cellobiose and
that gluconic acid is a significant inhibitor of the β-glucosidase.
Similar to the results of Dimarogona et al.,1025 Cannella and
colleagues demonstrate that the presence of lignin negates the
need to add external reducing agents.626 In a subsequent study,
Cannella and Jørgenson demonstrate that simultaneous
saccharification and fermentation conditions reduce the amount
of gluconic acid production likely due to oxygen uptake by the
fermentative organism.627 Lastly, the analysis of heterogeneous,
oxidized products from LPMOs has been a significant challenge
in the field that has required the development of new analytical
approaches. In a series of two papers, Isaksen et al. and
Westereng et al. report detailed spectroscopic and chromato-
graphic methods that enable chemically accurate and robust
methodologies for analysis of oxidized products.52,999
11.3. Conclusions
In terms of LPMO mechanisms of action, to date, we
collectively know the following:
(1) LPMO action requires a reducing agent and molecular
oxygen and a copper ion in the active site.48,50,51,981
(2) The reducing agent required for LPMO action can be
enzymatic (CDH), an externally added small molecule, or
lignin-derived molecules found in bio-
mass.48−51,312,626,965,967,979,995,997,999,1002,1024−1026
(3) LPMOs have been shown to definitively act either on C1
or C4 carbons,50,312,995,997,999 and several motifs have
been identified from biochemical and structural studies
that impart regioselectivity.997 Oxidative action at the C1
carbon yields a lactone, which can be hydrolyzed to an
aldonic acid motif, whereas C4 oxidation yields a 4-keto
sugar that can hydrolyze to a geminal diol.
(4) There have been three “auxiliary activity” families
characterized according to the CAZy database thus far
on the basis of sequence differences.152,1024 Different
LPMOs within the AA9 and 10 families can act either on
insoluble forms of cellulose or chitin and, in one reported
case to date, on soluble cello-oligomers,999 xyloglucan,1000
and starch.1001
(5) A theoretical prediction has been reported that suggests
the fungal LPMO mechanism employs a Cu(II)−oxyl
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ROS via an oxygen-rebound mechanism for polysacchar-
ide hydroxylation.1003
LPMOs are a recent discovery, and they are rapidly emerging
as a very important enzyme addition to the canon of cellulolytic
enzymes, which to date have primarily been hydrolytic. They are
also attracting interest from a fundamental perspective, given
their unique copper-binding sites and the fact that they catalyze
cleavage of very strong glycosidic bonds.1027 LPMOs are able to
synergize with GHs, likely as endo-acting enzymes that act
directly on the surface of crystalline polysaccharides. This ability
to act on crystalline regions offers a plausible explanation of the
apparent synergy of LPMOs with EGs, which are thought to
primarily act in more accessible, amorphous regions of cellulose.
As further evidence toward this hypothesis, in the case of α-
chitin, low crystallinity in the initial substrate reduces the
boosting potential of CBP21.1028
As discussed in section 2, glycosidic linkages in poly-
saccharides are among the strongest covalent linkages found
in nature.71 The hydrolytic enzymes that cleave these linkages
are thus some of the most powerful enzymes known, and the
discovery of LPMOs demonstrates that other enzymatic
paradigms are also able to selectively cleave these bonds.48
Transition metals are usually required in enzymes or oxidation
catalysts to circumvent spin-forbidden transitions in the triplet
ground state of molecular oxygen. The oxidative power required
to break strong bonds in carbohydrates coupled to the active site
geometries revealed in LPMO structures has drawn comparison
to methane monooxygenase, which activates one of the
strongest known C−H bonds.50,51,1002,1009 Similarly, Kim et al.
predicted that an incredibly powerful oxidative species, Cu(II)−
oxyl, is likely responsible for polysaccharide hydroxylation in a
fungal LPMO.1003
Going forward, there are undoubtedly many open questions
related to the mechanisms of these fascinating enzymes. In a
recent perspective on LPMOs,1002 Hemsworth, Davies, and
Walton posited that nonfungal (CBM33, AA10) and fungal
(GH61, AA9) LPMOs may employ a different catalytic
mechanism based on differences in the active site structures,
illustrated for representative structures in Figures 74 and 77, and
their EPR spectra. Specifically, nonfungal LPMOs examined to
date exhibit a conserved alanine residue in the active site that is
absent in fungal AA9 LPMO structures and sequence align-
ments. This alanine residue may alter the initial binding of
oxygen1003 or modulate the identity of the ROS.991,1002
Additionally, the identity of the axially coordinating residue
was thought for some time to be mainly tyrosine in fungal AA9
LPMOs and phenylalanine in nonfungal AA10 LPMOs, but it is
not known how universal this delineation is, or if the axial
residue plays a significant role in the reaction. In fact, Harris et
al. demonstrated in their initial study that the mutation of the
tyrosine residue to phenylalanine in a fungal LPMO reduced the
observed synergy, but did not remove it altogether suggesting
that the enzyme was still active.967 Later reports of AA10
LPMOs have shown that the axial residue can be tyrosine.1029
The report of the AA11 LPMO family was the first fungal
LPMO that exhibits an axial phenylalanine residue and an
alanine in the active site, in a seemingly intermediate active site
arrangement between AA9 and AA10 LPMOs.1024 Additionally,
there is a highly conserved residue in the active site that is
typically glutamine in fungal LPMOs and glutamic acid in
nonfungal LPMOs, but again its role in the oxidation reaction is
unknown. Additionally, the lack of N-methylation on the N-
terminal histidine in bacterially expressed AA10 LPMOs, or
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fungal LPMO heterologously expressed in yeast, suggests that 12. MODELING ENZYMATIC HYDROLYSIS
perhaps filamentous fungi are able to employ this post- Extremely valuable information regarding the catalytic and
translational modification whereas bacteria and some yeast processive mechanisms of CBHs and EGs can be obtained by
cannot. If this modification is significant for catalytic action, then considering each component in isolation. However, synergism
perhaps it might lead to differences in activity or even a different between multiple cellulase components cannot be captured by
mechanism. Lastly, X-ray radiation is able to rapidly photo- models that only consider one cellulase at a time. Kinetic models
reduce the copper ion from a copper(II) oxidation state to a of multiple enzyme components (cocktails) allow for testing of
copper(I) oxidation state in AA10 (and for at least one case, predictions and quantifying the effects of varying enzyme and
AA11) LPMOs. However, fungal LPMOs solved to date with substrate concentrations and properties.33,556 We review here
similar X-ray crystallography approaches are able to harbor two basic categories of kinetic models that have been applied to
copper(II) ions seemingly with no consideration for photo- the enzymatic hydrolysis of cellulose, namely ordinary differ-
reduction. This difference may suggest clues as to the differences ential equation (ODE)-based models and agent-based models.
in the mechanisms of these LPMO families. Regardless, In what follows, we first highlight ODE-based models (Figure
although the LPMO enzymes in the AA9−11 families are 79), followed by agent-based models (Figure 80).
distinctly separated in sequence space, it is not clear how
universal the active site differences are between the three LPMO
families, nor if this has significant ramifications on the oxidation
mechanism. Thus far, LPMO products and substrates do not
significantly differ between the families relative to the internal
variation per family. Undoubtedly, the more information that we
collectively elucidate regarding the mechanisms of these novel
enzymes will likely warrant constant evaluation as to their
classification. The differences in reactivity, now among the three
LPMO families,1024 will require considerable biochemical,
structural, and theoretical work to fully unravel.
Additionally, to date very little has been reported on the
electron transfer steps besides a study on the interaction of a
fungal LPMO with two different CDHs.1026 Sygmund et al.
examined the effect of the two native CDHs from N. crassa in
their interaction with an LPMO from the same organism, and
found that there are differences in the efficiency of CDH/
LPMO interaction, but the authors point out that this requires a
significantly expanded scope to be able to ascertain LPMO−
CDH intermolecular interactions and catalytic efficiencies.1026
Intramolecular electron transfer in LPMOs has been postulated
to occur via clusters of aromatic residues,991,995 but the test of
this hypothesis has not yet been reported. The need for further
studies into the interactions of CDH or small-molecule reducing
agents with LPMOs is obvious, and may be appropriate for
biophysical studies such as those utilizing surface plasmon
resonance, NMR, or even cocrystallization.
Beyond the detailed, molecular mechanisms of LPMO action,
Figure 79. Illustrative example of an ODE-based model for the
there are many questions that need to be answered related to
enzymatic degradation of crystalline cellulose. (A) An example of the
industrial applications of LPMOs. The design of optimal discrete steps of CBH processive/hydrolytic activity that may be
mixtures of GHs and LPMOs will be required for biomass captured by an ODE based kinetic model. Kinetic schemes to capture
depolymerization in a biofuels or chemicals context, and this the CBH processive action may assume (B) that CBH can only
work will require massive efforts similar to the decades of efforts produce cellobiose or (C) that initial cuts produce glucose, cellobiose,
already spent optimizing GH cocktails and understanding and cellotriose with nonzero probability. Note that the cycle in part A
enzyme synergy. The application of sophisticated enzymology does not correspond precisely to either of the schemes shown in parts B
tools such as those reviewed above from Väljamäe et or C. Part A is reprinted with permission from ref 532. Copyright 2011
American Chemical Society. Parts B and C are reprinted with
al.,307,531,557 Westh et al.,559,560 and Igarashi et al.476,558 will
permission from ref 1040. Copyright 2011 Wiley Periodicals, Inc.
undoubtedly help elucidate the mechanisms of synergy in a
more detailed fashion, which will accelerate the development of
optimized GH/LPMO cocktails. 12.1. Ordinary Differential Equation-Based Models
Lastly, the discovery of LPMOs 20 years after the first ODE-based kinetic models first formulate a set of process steps
structures of the first GH enzymes from fungi172,173,192 and 60 (Figure 79A) and then develop a set of differential equations
years after the C1−Cx hypothesis was put forth by Reese et that describe these steps (Figure 79B,C). The rate constants
al.291 highlights the fact that we still have much to learn about necessary for each process step generally come from experi-
novel biomass depolymerization mechanisms in nature. There ments (or sometimes from simulations) wherein the kinetics of
are likely more discoveries on the level of LPMOs that we still individual steps have been isolated. Varying system inputs (e.g.,
have yet to uncover about how fungi and organisms from other starting concentrations of substrate or enzyme, substrate
kingdoms of life degrade plant cell walls. properties, the reaction rate constants, etc.) and solving the
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Figure 80. Illustrative example of an agent-based model for crystalline
cellulose enzymatic degradation. (A) At the beginning of an
adsorption/hydrolysis simulation the substrate surface lattice is
perfectly ordered. (B) After adsorption equilibrium has been reached,
nonproductively bound CBHs contribute to a reduced apparent
processivity. (C) Surface erosion is exemplified after limited hydrolysis
and (D) after extended hydrolysis. Reprinted with permission from ref
600. Copyright 2001 John Wiley and Sons.
resulting set of differential equations can reveal the dependency
of various outputs (product concentrations, conversion, degree
of synergism, etc.) on the inputs.
In the earliest model of this type, Suga et al. predicted the
molecular weight distribution of saccharides released from an
insoluble substrate, considering EG and CBH (the latter acting
exclusively at chain ends) both in isolation and in concert.1030
They modeled the substrate−enzyme interaction as Michaelis−
Menten kinetics, but this is solely for the reaction and not for
adsorption, which they did not consider. They assumed that all
β-glycosidic bonds are accessible to the enzymes. They noted
that, with EG alone, the production of monomers (in this case,
cellobiose) is extremely low due to its random cleavage of
internal bonds. On the other hand, CBHs produce only
monomers, but have a low concentration of chain-end glycosidic
bonds available to cleave. In concert, however, they demon-
strated that the monomer production was drastically increased,
thus capturing endo/exo synergism.1030
The model of Okazaki et al. added to the approach of Suga et
al. the inclusion of β-glucosidase activity as well as consideration
of product inhibition.1031 Their results predicted that CBH
catalytic activity is inversely proportional to the initial substrate
DP and that EG activity is not dependent on DP. However, the
synergistic effect of employing EG and CBH in concert actually
increases as the substrate DP increases.1031
Converse and Optekar considered a binary cocktail of EG and
CBH1032 in order to rationalize the fact that the degree of
synergism had been shown1033 to go through a maximum as the
total enzyme concentration increased. Their model featured
competitive adsorption of the two cellulases for a limited
number of sites. At low enzyme concentrations, the system is
also at low conversion, and the CBH is less dependent on the
new chain ends created by the EG. At high enzyme
concentrations, CBH dominates the surface sites and thus
restricts the EG from creating new chain ends. An optimal
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enzyme loading exists in between these two limits, resulting in a
maximum degree of synergy.1032
Eriksson et al. modeled the synergistic action of TrCel7A and
TrCel7B (a CBH and an EG, respectively) on steam-pretreated
spruce and attempted to account for the rapid rate decrease seen
in enzymatic hydrolysis.569 Their experiments indeed exhibited
this rate decline, and they were able to rule out enzyme thermal
stability and product inhibition as major causes. Addition of
TrCel7B increases the hydrolysis rate more than adding
TrCel7A, which in turn has a greater effect than adding
substrate. Their explanation for EG/CBH synergism centered
on the ability for EGs to “remove” disordered cellulose chains
that act as obstacles to processive CBH action. This proposal has
gained a lot of traction in recent years as convincing
experimental evidence has pointed to steric obstacles as a rate-
limiting factor for CBHs acting in isolation307 and their removal
as a vital role for EGs.557
Zhang and Lynd developed a model for an enzyme cocktail
with parameters chosen from the experimental literature for
TrCel7A, TrCel6A, and TrCel7B.1034 Notably, they apply a
single set of enzyme parameters to various “substrates” (by
examining characteristic values of degree of polymerization DP
and fraction of β-glucosidic bonds available to cellulase). They
conclude that these two substrate parameters are sufficient to
capture various phenomena such as the dependence of cellulase
synergy on the substrate properties, enzyme loading, and
reaction time. In addition, of the two substrate properties, they
predict that enhancing substrate availability is more beneficial to
increasing activity than decreasing DP.1034
Bommarius et al.596 compared two approaches to the problem
of enzyme adsorption, diffusion, and reaction, namely fractal
kinetics and jamming kinetics. Fractal kinetics accounts for the
heterogeneous nature of the substrate that restricts cellulase
diffusion, whereas jamming kinetics focuses on the obstruction
of cellulase motion due to other bound cellulases. Exper-
imentally, they studied the effect of using an array of different
pretreatments on rate slowdown. They find that the rate of
hydrolysis dropped by 2−3 orders of magnitude at high degrees
of conversions, but this is not dependent on the pretreatment
method. In fact, they conclude that pretreatment and fractal
kinetics are irrelevant to the rate decline seen at high degrees of
conversion. Their model explained the decline, however, as
being due to the jamming of enzymes bound to the substrate
surface.596
Zhou et al. introduced a model that explicitly accounted for
the changing morphology of the substrate due to enzymatic
hydrolysis as well as the cellulose chain fragmentation.1035−1037
This is an important concept, as the evolution of accessible
surface area is likely to be a complex function of multiple
dynamic parameters throughout the course of hydrolysis. This
model was applied to the T. reesei system of Cel7A, Cel6A, and
Cel7B degrading Avicel,1035 and they were able to explicitly
capture the hydrolytic evolution of cellulose substrate. Their
results indicated that, in addition to averaged substrate
characteristics such as DP, internal surface area, and enzyme
accessibility fraction, that the distribution of structural
heterogeneities is an important characteristic to be considered.
Their model captured the rapid rate retardation of enzymatic
hydrolysis, and the authors proposed that this distribution is an
important contributing factor in the slowdown. Their “surface
ablation” model is conceptually similar to the lattice models of
Sild et al.600,1038 discussed below. Within this model, there are
two relevant substrate-related time scales: the time to degrade a
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single glucan chain (nonmorphological) and the time to
completely degrade the substrate (which necessitates morpho-
logical consideration). The model is able to correlate the first
time scale with the initial rapid rate slowdown,1037 a finding that
is compatible with the steric obstacles hypothesis.307 However,
the model is not able to match experiments in regard to the
second time scale, leading to the conclusion that other inhibitory
factors must be at work in this regime.1037
Levine et al. introduced a mechanistic model that included the
following novel features: enzyme adsorption and complexation
were modeled as distinct steps, the equilibrium assumption for
adsorption was abandoned, and representation of cellulosic
substrate was as a polydisperse distribution of spheres.1039 They
applied this to the individual and synergistic behavior of Cel7A
and Cel5A from T. reesei. Their model performed well except
with low surface area, where enzyme competition for adsorption
sites is particularly high and enzyme crowding is most relevant.
Also, the experimentally observed retardation of hydrolysis
could only be accounted for with abnormally strong product
inhibition or short enzyme half-lives; thus, they concluded that
neither of these were the primary causes of the slowdown. They
also found that enzyme crowding cannot be the sole reason for
the hydrolytic rate slowdown; model results only showed this
effect with multiple enzymes present and with high substrate
loadings, whereas experiments find the rate slowdown to be
universal (even with single enzymes). They note that “structural
heterogeneities” may play a significant role, but their model did
not account for these.
Levine et al. later expanded their previous model to track
individual product formation (glucose, cellobiose, and cello-
triose) instead of just overall hydrolytic activity,1040 with the
goal of developing a rational method for determining the
optimum cellulase ratios for hydrolysis experiments (using
Cel7A, Cel6A, and Cel5A from T. reesei). They considered
various combinations of substrate DP and surface area, including
values representative of BMCC. The model results suggested
that optimal enzyme ratios are 1:0:1 with Cel7B:Cel7A:Cel6A at
shorter times (24 h) and 1:1:0 at longer times (72 h); the
authors indicated this may be related to the relative thermal
stabilities of the two CBHs.
Informed by the kinetic models just described, Fox et al.
experimentally examined the kinetics of CBH chain complex-
ation and how EGs affect these kinetics.532 They employ long-
time hydrolysis trials (>100 h) of BMCC by CBH Cel7A from
T. longibrachiatum and EG II from T. emersonii. They measured
initial-cut product release and equated this to initial chain
complexation. They also measured the processive-cut products
and deduced processivity from the ratio of processive-cut
products to initial-cut products. Via material balances on the
initial-cut products and the processive-cut products, they
developed a system of ODEs. Even when chain-ends for CBH
action were in excess (due to high concentration of EG), the rate
of generation of initial-cut products was an order of magnitude
lower than the hydrolysis rate of soluble cellohexaose. From this
and other evidence, it was concluded that chain complexation is
rate-limiting. This was in contrast to the findings of Kurašin and
Väljamäe.307 However, Fox et al. also found that increasing EG
concentration decreases the length of a processive run for the
CBH while also increasing the hydrolytic conversion. This
finding perhaps supports the idea that EGs help to “rescue”
CBHs that are trapped behind obstacles.557 This would explain
the trends seen in processivity and conversion within the
obstacle-limiting framework, though it does not fit as well in the
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complexation-limited framework. Fox et al.’s calculation for
apparent processivity was much lower than for intrinsic
processivity; thus, they concluded that CBHs do have their
processive runs halted by steric obstacles on the surface,532
consistent with Kurašin and Väljamäe.307
Griggs et al. developed a population balance model to capture
the hydrolysis of cellulose by action of an EG and CBH.1041 For
the CBH, distinct steps for the adsorption, complexation,
processive hydrolysis, and desorption were modeled. They
captured temporal development of the chain length distribution
for EG in isolation, CBH in isolation, and EG/CBH in concert
for both amorphous and structured substrate. In a follow-up
study, β-glucosidase action was included to model a complete
cocktail. The rate-limiting factor in this study was determined to
be the availability of substrate.1042
One of the most difficult aspects of gaining mechanistic
insight into how cellulases work is that surface interactions are
very difficult to characterize.1043 Fox et al. utilized photo-
activated localization microscopy (PALM), and applied it to
quantify the binding affinity of six different CBMs for regions of
a cellulosic cotton substrate of varying crystallinity.1044 Their
results demonstrated quantitatively the invalidity of applying a
purely binary classification to cellulose (amorphous vs
crystalline). They quantitatively assessed the binding preference
of the different CBMs studied and found a continuum of
binding preferences. They then related these findings to binary
synergistic enzyme activity by synthesizing chimeras composed
of A. cellulolyticus GH5 EG, TrCel7A linker, and each of the six
CBMs. Their results revealed a significant new element of
cellulase synergism. If two cellulases worked in either totally
different regions of cellulose or in the same region, there is no
synergistic effect. However, enzymes with similar but non-
identical binding site preferences gave optimal synergism, by
enhancing the susceptibility of the substrate for one another.
These conclusions have clear implications for selecting enzymes
for cocktails with optimal synergism.
Gao et al. challenged the conventional paradigm that
increased hydrolytic activity positively correlates with an
increase in surface-bound enzyme.1045 With a cocktail of
TrCel7A, TrCel6A, and TrCel7B, they showed that although
the binding coefficient on cellulose III was only half that on
native cellulose Iβ, its hydrolytic activity was increased. In fact,
they showed that enzyme loading can be decreased by 5-fold on
cellulose III to achieve concomitant hydrolysis rates as on
cellulose Iβ. To rationalize this observation, they developed a
kinetic model that couples enzyme binding, chain decrystalliza-
tion/sliding, and hydrolysis as distinct steps in the enzymatic
processive cycle. Their model showed that their nonintuitive
experimental result can be reproduced if the enzymes’
processive ability (quantified by rate constant kslide) was
enhanced while its initial chain-association ability (quantified
by nkon, where n is the number of available binding sites and kon
is the adsorption rate constant) was diminished.
12.2. Agent-Based Models
Agent-based models (also known as cellular automata) treat the
cellulosic substrate and enzymes as individual entities and assign
behaviors or properties to each on the basis of previously
published parameters. They have the advantage of giving a
spatial dimension to traditional models that are solely based on
differential equations. In addition, they provide the additional
capability of visual inspection, which can give insight into
mechanisms and guide further model development (Figure 80).
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Finally, this type of model more naturally captures physical
phenomena on the substrate surface such as enzyme crowding
and nonproductive binding to substrate.
Sild et al. employed an infinite, anisotropic, two-dimensional
lattice simulation with overlapping binding sites (of only one
type) with the goal of estimating the amount of bound enzyme
as a function of free enzyme.1038 Simple rate equations governed
the binding and dissociation of CBH (e.g., Cel7A) to/from an
initially perfect crystalline surface (e.g., Avicel). Though
experimentally it had been shown that the adsorption of
TrCel7A to cellulose could be accounted for by assuming two
different types of binding sites (perhaps signifying regions of
high and low crystallinity), Sild et al. demonstrated with their
model that if the binding sites are allowed to overlap (as is the
case experimentally),348 the data is fit equally well by a model
that assumes only one type of binding site. Their data also
indicate that, at high cellulase loadings, the surface reaches an
apparent equilibrium before the theoretical maximum coverage
due to surface crowding and slow rearrangement. This resulted
in surface coverage that is as much as 40% below the theoretical
maximum, depending on the shape of the adsorbing molecule.
Later, Väljamäe et al.600 coupled experiments with an
expanded version of the Monte Carlo simulation model of
Sild et al.1038 by adding two significant aspects: TrCel6A was
included (in addition to TrCel7A), and the hydrolysis, as well as
the binding, was considered. Experimentally, they find hydro-
lytic rate retardation at short times (in the first 5−10 min). Also,
preincubation of BMCC with TrCel7A actually decreased the
reaction velocity for subsequent hydrolysis with TrCel7A.
However, preincubation with TrCel6A increased the reaction
velocity for subsequent hydrolysis by TrCel7A. Thus, so-called
exo/exo synergism was not dependent on the CBHs being
incubated simultaneously. They subsequently sought to explain
these results via Monte Carlo simulations. They attributed the
initial rate retardation to two primary factors: steric hindrance
by nonproductively bound cellulases and cellulose surface
erosion due to CBH action. Their simulations support these two
modes of CBH inhibition (Figure 80). The early stages of
adsorption/desorption simulations featured the most drastic
increase in bound CBHs causing a concomitant increase in
enzyme obstacles. In addition, CBH processive action degraded
the surface from an initially homogeneous one to a drastically
eroded one; at long times, a steady-state erosion pattern having
a constant retarding effect should be established.600
Fenske et al. examined the case of a single cellulase that has
nonzero exo and endo capabilities acting on an insoluble
polysaccharide substrate, modeled as a two-dimensional lattice
of fixed DP.1046 Enzymes were allowed to move and cleave
bonds on the surface in a stochastic fashion (with fixed
probabilities). Their results indicated that substrate inhibition
was present at high substrate loadings with only one cellulase
component. This was due to a decrease in “autosynergism”, that
is, the endo activity of an enzyme aiding the exo-activity of the
same enzyme.1046
Warden et al. developed a cellular automata model to model
the deconstruction of cellulose to glucose by cellulase
cocktails.1047 They developed a three-dimensional spatial lattice
model that included control over many physical and chemical
variables involved in the saccharification of cellulose. They
utilized published values for the cellulase system for T. reesei
(section 4.3) and examined the effect on glucose production of
varying enzyme loadings, adsorption strengths, and catalytic
activities of the EGs and CBHs in the system. In general, they
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Review
found that varying the enzyme/substrate ratio and adsorption
strength (for either CBH or EG) from the literature values
resulted in a decrease in glucose production. They found
decreasing glucose production with time and attributed this to
enzyme crowding on the surface and decreased cellulose surface
area.
Asztalos et al. utilized a spatial model for cellulose degradation
that “forms a bridge” between all-atom MD simulation and
higher-level ODE models that accounted for enzyme crowding
on the surface.1048 They developed a coarse-grained stochastic
model of endo and exo cellulases with a two-dimensional surface
modeling crystalline cellulose. They examined TrCel7A,
TrCel6A, and TrCel7B and include the following reactive
events: adsorption, interchain hydrogen bond breaking,
hydrolysis of glycosidic bonds, and desorption. Endo/exo
synergism was qualitatively reproduced.1048
In summary, both ODE- and agent-based models of cellulose
degradation by enzyme cocktails have supplemented and
improved experimental characterization of the substrate and
enzymatic action. In particular, they have shed light on the
mechanisms and influencing factors of significant experimentally
observed phenomena including cellulase synergism and the
rapid initial retardation in hydrolysis rate. These models offer
the ability to test mechanistic explanations for these phenomena
as well as to strip the system down to its basic essentials in order
to offer these insights. As such, they can inform the selection of
process conditions, such as optimal cellulase loadings and ratios
between cocktail components. Finally, they have the potential of
assisting in the determination of the relative rates of the
individual steps of the processive cycle of CBHs. Going forward,
these models have the potential to enhance mechanistic
understanding, particularly as they are increasingly coupled to
advanced experimental techniques (refs 102, 103, 128, 307, 476,
521, 532, 534, 535, 546, 548, 552, 557, 558, 560, 561, 564, 795,
and 1044) and include the synergistic action of CBHs (with
endo and exo capability and acting from both reducing-end and
nonreducing-end), EGs, and LPMOs.
13. CONCLUDING REMARKS
Fungi have evolved to be the most powerful and prevalent
biomass degrading organisms in nature, exhibiting a diverse
range of lifestyles for the turnover of lignocellulosic material on
Earth. Given their significant activity and ability to be readily
produced at high titers on the industrial scale, fungal cellulase
cocktails are an excellent starting point for industrial biorefinery
applications. Indeed, industrial-scale lignocellulosic biorefi-
neries, mostly slated to produce bioethanol at this point, are
beginning to come online worldwide at the time of this review.
Given the scale of fuel demand and the significant potential for
biomass conversion to offset fossil fuel usage, the use of fungal
cellulases will likely grow dramatically in the coming years,
which in turn could make fungal cellulases the most widely
produced industrial enzymes in the world by a large margin. As
enzymes remain a major cost in renewable fuels production, this
in turn makes even “small” gains in enzyme or cocktail
performance of significant importance in the renewable energy
economy.
As covered in this review, significant strides in our
fundamental understanding of cellulase action have been made
in the past several decades, especially driven by the structural
biology efforts starting in the early 1990s. However, challenges
to “well-accepted” models and the basic physical mechanisms of
cellulose deconstruction have been reported in the last several
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years that highlight how much we have yet to understand. For industrial world. Advances from the worldwide research
example, the role of hydrolytic EGs has been recently revealed community will be needed to comprehensively address these
to be generation of “dissociation” points for CBHs, instead of open questions and to harness the considerable potential of
the commonly cited attachment points (see section 6.2). these fascinating enzymes.
Additionally, the recent discovery of LPMOs highlights the fact
that basic cellulolytic paradigms are not fully characterized, and AUTHOR INFORMATION
it is possible that other enzyme activities exist that have not yet
been discovered, or like LPMOs, may be misclassified. Beyond Corresponding Author
those two examples, many open questions remain even *E-mail: gregg.beckham@nrel.gov.
regarding basic catalytic mechanisms in some cases (e.g., GH6
Author Contributions
cellulases and LPMOs), the molecular basis for activity
#
differences within the same family of cellulolytic enzymes, the These authors contributed equally to the review.
basis of synergistic function in enzyme cocktails (e.g., why are Notes
multiple EGs needed? What function do LPMOs serve beyond
EGs?), and enzyme−substrate interactions across the multiple The authors declare no competing financial interest.
length scales likely of importance in cellulose (and biomass) Biographies
deconstruction. Unlike most enzymes that work in solution,
cellulases function at a solid−liquid interface on a physically
heterogeneous substrate in the case of clean cellulose and
physically and chemically heterogeneous substrate in the case of
native or pretreated plant cell walls. Our lack of understanding
of these solid substrates coupled to the inherent difficulties
associated with effective kinetics measurements of single
enzymes and enzyme cocktails makes the challenge of
understanding and improving cellulase action a daunting
technical challenge. Moreover, for both fundamental inves-
tigations and industrial applications, the substrate of choice must
be carefully considered for interpretation of results, and in the
latter case of pretreated biomass, the substrate treatment must
be “co-optimized” with the enzyme cocktail design. Taken
together, these recent findings and open questions continue to
challenge our collective basic premise of cellulose deconstruc- Christina M. Payne is an assistant professor in the Department of
tion and highlight the fact that exciting, impactful advances are Chemical and Materials Engineering at the University of Kentucky and
yet to be made in the study of cellulose deconstruction in both the August T. Larsson Guest Researcher at the Swedish University of
natural and applied contexts. Agricultural Sciences in Uppsala, Sweden. She received her Ph.D. in
Going forward, these open questions will be addressed chemical engineering from Vanderbilt University in 2007. After a brief
through the continued application of traditional structural foray into the world of chemical process engineering, she undertook
biology and biochemical measurements, development and postdoctoral studies at the National Renewable Energy Laboratory
application of advanced biophysical measurements, kinetic and (NREL) in 2011 and was promoted to staff scientist the same year. In
molecular modeling coupled to experimental findings, screening 2012, she joined the faculty at the University of Kentucky where her
and mining natural diversity of both natural enzymes and research focuses on application of computational tools to investigate
secretomes, detailed substrate characterization, and the develop- protein structure−function relationships and biocatalysis.
ment of novel methods for engineering enzymes that inherently
work at solid−liquid interfaces in a cocktail context. The
elucidation of rate-limiting steps in “simple” cocktails via
advanced methodologies developed in the last several years
has begun to uncover some aspects of cellulolytic action that can
be considered targets for enzyme engineering. Continued
development and application of these types of quantitative
methods in increasingly complex systems and across substrates
will further elucidate the physical and chemical mechanisms of
cellulase action, and highlight opportunities for improvement of
enzyme performance.
In conclusion, fungal cellulases, some of which remain to be
discovered, work in concert to accomplish one of the most
important processes in nature, namely the turnover of
recalcitrant cellulose. This process is of paramount importance
in the global carbon cycle and may become one of the most Brandon C. Knott is a Postdoctoral Research Fellow in the National
industrially important enzymatic reactions given the desperately Bioenergy Center at NREL and recipient of the NREL Director’s
needed drive toward a renewable energy-based global society. Fellowship. He obtained his Ph.D. from the University of California,
Understanding the function, diversity, and mechanisms of fungal Santa Barbara, in 2012 in Chemical Engineering for research into the
cellulases remains an extremely challenging, massive endeavor mechanisms of nucleation from solution utilizing statistical mechanics,
but one of considerable importance for both the natural and computer simulation, and experiments. His current research at NREL

1429 DOI: 10.1021/cr500351c

Chem. Rev. 2015, 115, 1308−1448
Chemical Reviews Review

focuses on utilizing advanced computational approaches to elucidate

mechanisms of enzymatic catalysis relevant to biofuels production.

Michael E. Himmel is a Research Fellow in the Biosciences Center at

NREL. He obtained his Ph.D. from Colorado State University in 1980
in Biochemistry. At NREL, he has conducted, led, and designed
Heather B. Mayes is a Ph.D. candidate in the Department of Chemical research in protein biochemistry, recombinant technology, enzyme
engineering, and new microorganism discovery. During the past three
and Biological Engineering at Northwestern University in Evanston, IL,
decades, he has contributed over 300 journal articles, 7 books, and 22
where she is using computational chemistry to elucidate thermochem- patents to the literature. In 2004, he was a recipient of an R&D 100
award for “Advanced Catalytic System for Biomass Conversion”.
ical and enzymatic routes for cellulose decomposition to produce
renewable fuels and chemicals. She is a Department of Energy
Computational Science Graduate Fellow, Chicago ARCS Scholar, and
active member of the Society of Women Engineers. Before returning to
school, she worked as a chemical engineering consultant for Jacobs
Consultancy, helping transportation fuel companies evaluate potential
new processes and make existing processes safer and more energy-
efficient.

Mats Sandgren is an Associate Professor at the Department of

Chemistry and Biotechnology, Swedish University of Agricultural
Sciences, Uppsala, Sweden, and has studied cellulose degrading
enzymes since 1998. At Uppsala University he obtained his Ph.D. in
molecular biology in 2003, in the group of Alwyn Jones, for research on
structure−function relationships in cellulases. He and his research
group moved to the Swedish University of Agricultural Sciences in
2007 and focus now on research on structure−function relationships in
cellulases and other carbohydrate-active enzymes.

Henrik Hansson is a Researcher at the Department of Chemistry and

Biotechnology, Swedish University of Agricultural Sciences, Uppsala,
Sweden. He obtained his Ph.D. from the Royal Institute of Technology,
Stockholm, in 2002 for research in protein−protein interaction using
NMR and other biophysical techniques. A postdoctoral period followed
at Uppsala University, Uppsala, Sweden, working with Dr. Gerard J.
Kleywegt in the laboratory of Professor T. Alwyn Jones. In 2006, he
went to the Swedish University of Agricultural Sciences and
Department of Molecular Biology, now the Department of Chemistry
and Biotechnology, to use crystallography and structural biology to
Jerry Ståhlberg is an Associate Professor at the Department of
study cellulases and other cellulose degrading enzymes. Chemistry and Biotechnology, Swedish University of Agricultural

1430 DOI: 10.1021/cr500351c

Chem. Rev. 2015, 115, 1308−1448
Chemical Reviews Review

Sciences, Uppsala, Sweden, and has studied cellulose-degrading EPR electron paramagnetic resonance
enzymes since the mid-1980s. At Uppsala University he obtained his GH glycoside hydrolase
Ph.D. in biochemistry with Göran Pettersson, and he then joined the GH5 family 5 glycoside hydrolase
structural biology group of Alwyn Jones. He moved to the Swedish GH6 family 6 glycoside hydrolase
University of Agricultural Sciences in 1998 and is leading research on GH7 family 7 glycoside hydrolase
structure−function relationships in cellulases and other carbohydrate- GH12 family 12 glycoside hydrolase
active enzymes. GH45 family 45 glycoside hydrolase
GH61 family 61 glycoside hydrolase
Glc3 cellotriose
Glc4 cellotetraose
Glc5 cellopentaose
Glc6 cellohexaose
Glc8 cellooctaose
Glc9 cellononaose
LPMO lytic polysaccharide monooxygenase
MCC microcrystalline cellulose
MD molecular dynamics
NMR nuclear magnetic resonance
PASC phosphoric acid swollen cellulose
oNPC o-nitrophenyl-β-D-cellobioside
pNPC p-nitrophenyl-β-D-cellobioside
pNPL p-nitrophenol-β-D-lactoside
Gregg T. Beckham is a Senior Engineer in the National Bioenergy RMSD root-mean-square deviation
Center at NREL in Golden, Colorado. He obtained his Ph.D. from the SAXS small-angle X-ray scattering
Massachusetts Institute of Technology in 2007 in Chemical Engineer- SEM scanning electron microscopy
ing for research into solid-state nucleation mechanisms using statistical TEM transmission electron microscopy
mechanics and molecular simulation. After joining NREL in 2008, his XRD X-ray diffraction
research interests have focused on understanding structure−function
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1448 DOI: 10.1021/cr500351c

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