doi:10.1016/j.jmb.2011.02.028 J. Mol. Biol.

(2011) 408, 177–186

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Journal of Molecular Biology

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COMMUNICATION

Crystal Structure of Glucansucrase from the Dental

Caries Pathogen Streptococcus mutans
Keisuke Ito 1 †, Sohei Ito 1 ⁎†, Tatsuro Shimamura 2,3 †,
Simone Weyand 4,5 , Yasuaki Kawarasaki 1 , Takumi Misaka 6 ,
Keiko Abe 6 , Takuya Kobayashi 2,3 , Alexander D. Cameron 4,5
and So Iwata 2,3,4,5
1
Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences,
University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka 422-8526, Japan
2
Japan Science and Technology Agency, ERATO, Iwata Human Receptor Crystallography Project, Yoshida-Konoe-
cho, Sakyo-ku, Kyoto 606-8501, Japan
3
Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto,
606-8501, Japan
4
Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London
SW7 2AZ, UK
5
Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot,
Oxfordshire OX11 0DE, UK
6
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of
Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8567, Japan

Received 8 November 2010; Glucansucrase (GSase) from Streptococcus mutans is an essential agent in
received in revised form dental caries pathogenesis. Here, we report the crystal structure of S. mutans
3 February 2011; glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble
accepted 8 February 2011 glucans and is a glycoside hydrolase (GH) family 70 GSase in the free
Available online enzyme form and in complex with acarbose and maltose. Resolution of the
25 February 2011 GTF-SI structure confirmed that the domain order of GTF-SI is circularly
permuted as compared to that of GH family 13 α-amylases. As a result,
Edited by G. Schulz domains A, B and IV of GTF-SI are each composed of two separate
polypeptide chains. Structural comparison of GTF-SI and amylosucrase,
Keywords: which is closely related to GH family 13 amylases, indicated that the two
Streptococcus mutans; enzymes share a similar transglycosylation mechanism via a glycosyl-
glucansucrase; enzyme intermediate in subsite − 1. On the other hand, novel structural
dental caries; features were revealed in subsites + 1 and + 2 of GTF-SI. Trp517 provided
glycoside hydrolase the platform for glycosyl acceptor binding, while Tyr430, Asn481 and
family 70; Ser589, which are conserved in family 70 enzymes but not in family 13
circularly permutation enzymes, comprised subsite + 1. Based on the structure of GTF-SI and amino
acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared
to be the most critical point for acceptor sugar orientation, influencing the

*Corresponding author. Laboratory of Food Protein Engineering, University of Shizuoka, Yada 52-1, Suruga-ku,
Shizuoka 422-8526, Japan. E-mail address: itosohei@u-shizuoka-ken.ac.jp.
† K.I., S.I. and T.S. contributed equally to this work.
Abbreviations used: ASase, amylosucrase; BLA, Bacillus licheniformis α-amylase; GH, glycoside hydrolase; GSase,
glucansucrase; GTF, glycosyltransferase; PDB, Protein Data Bank.

0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.
178
transglycosylation specificity of GSases, that is, whether they produced
insoluble glucan with α(1–3) glycosidic linkages or soluble glucan with α(1–6)
linkages. The structural information derived from the current study should be
extremely useful in the design of novel inhibitors that prevent the biofilm
formation by GTF-SI.
Sweet is an important favorable taste quality
linked to food intake in humans. Sucrose is the
most highly consumed sweetener but also frequent-
ly causes dental caries.1 According to the World
Oral Health Report 2003 released by the World
Health Organization, dental caries is a major health
problem in most industrialized countries, affecting
60–90% of school children and the vast majority of
adults. If left untreated for a long period of time,
dental caries, viewed as a “life-style-related disease,”
can result in pain, tooth loss, infection and, in some
cases, even death by sepsis.
Streptococcus mutans is the pathogen that is most
closely associated with dental caries.2 Caries forma-
tion is initiated when glucan, a sticky glucose
polymer produced by S. mutans, forms a biofilm
(or dental plaque) on teeth, which then traps oral
bacteria, food debris and salivary components.
Acids produced by bacteria in the biofilm as a result
of fermentation of dietary carbohydrates such as
sucrose, fructose and glucose demineralize the tooth
surface, leading to dental caries. High-molecular-
weight sticky glucan synthesized from sucrose by
glucansucrases (GSases), which are extracellular
enzymes expressed by S. mutans, plays an essential
role in the etiology and pathogenesis of dental
caries.3 Sticky glucan adheres to tooth surfaces to
form dental plaques, which can in turn cause
additional infections, periodontal disease and
halitosis.4 GSases are members of glycoside hydro-
lase (GH) family 70.2 They catalyze the formation of
glucan with various types of glucosidic linkages,
namely, α(1–3), α(1–4) or α(1–6) bonds, from
sucrose via transglucosylation reactions. GSases
are classified into four different types based on the
nature of the glycosidic linkage they catalyze:
mutansucrase, dextransucrase, alternansucrase and
reuteransucrase, which catalyze α(1–3), α(1–6), α(1–3
and 1–6) and α(1–4 and 1–6) glycosidic linkages,
respectively.5 In oral cavities, sticky glucan synthesis
by S. mutans involves three extracellular glycosyl-
transferases (GTFs): GTF-I, GTF-SI and GTF-S.6
GTF-I and GTF-SI are mutansucrases that synthesize
mainly insoluble glucan with α(1–3) glycosidic
linkages. GTF-S is a dextransucrase that synthesizes
predominantly soluble glucan with α(1–6) linkages.5
GSases have a molecular mass of approximately
160 kDa and are composed of three functional
regions: the N-terminal variable junction region, the
catalytic region and the C-terminal glucan binding
Crystal Structure of Glucansucrase
© 2011 Elsevier Ltd. All rights reserved.
region.5,7 The highly conserved catalytic region is
essential for glucan synthesis by GSases.
Several inhibitors of S. mutans GSase have been
identified.8–10 Structural information on this class of
enzyme could facilitate further development of
novel inhibitors of GSases that prevent dental caries
formation; however, to date, this type of information
has been lacking. Here, we present the crystal
structure of GTF-SI, a GH family 70 mutansucrase
that synthesizes mainly α(1–3)-linked insoluble
glucan. We also report the structures of GTF-SI in
complex with the inhibitor acarbose and acceptor
maltose. These structures provide critical insight
into glucan formation by this type of enzyme.
Overall structure of GTF-SI
Recombinant and selenomethionine-substituted
GTF-SI from S. mutans was expressed, purified and
crystallized.11 The detailed experimental procedure
for each structure is provided in Supporting
Information Materials and Methods. The final
refinement statistics for all structures are summa-
rized in Table 1. The monomeric structure of
substrate-free GTF-SI is shown in Fig. 1a. While
GTF-SI formed a tetramer in the crystal (Fig. S1a),
the results of size-exclusion chromatography indi-
cated that GTF-SI is likely a monomer in solution
(Fig. S1b), and the structures of the individual
tetramer members were almost identical. The
current model, refined at 2.1 Å resolution, contains
amino acid residues 244–1087 because the crystalli-
zation was not succeeded from the entire protein of
GTF-SI. The N-terminal His-tag and some of the
C-terminal residues (amino acid residues 1088–1163)
included in the current expression construct were
not observed in the electron density map. The
secondary structure of GTF-SI is shown schemati-
cally in Fig. S2. The structure of GTF-SI comprised
four separate domains: A, B, C and IV (Fig. 1a). Each
domain, with the exception of domain C, was
composed of two separate regions of the polypeptide
chain. Domain IV consisted of IV1 (residues 244–372)
and IV2 (residues 1057–1087); domain A consisted of
A1 (residues 443–692) and A2 (residues 829–961); and
domain B consisted of B1 (residues 373–442) and B2
(residues 962–1056). Domain C was composed of a
single stretch of amino acid residues (693–828).
GH family 13 amylases have high level of
similarity in overall structures, especially in domain
Crystal Structure of Glucansucrase 179

Table 1. Crystallographic statistics

Selenomethionine peak Wild type Maltose Acarbose
Data collection
Space group P21212 P21212 P21212 P21212
Cell dimensions (Å) a = 295.43 a = 293.88 a = 295.42 a = 295.52
b = 214.50 b = 215.42 b = 213.94 b = 214.41
c = 219.52 c = 218.95 c = 220.98 c = 220.66
Wavelength (Å) 0.9792 1.0000 0.9796 0.9796
Resolutiona (Å) 30.00–2.70 (2.80–2.70) 50.00–2.10 (2.18–2.10) 50.67–3.09 (3.27–3.09) 61.77–3.11 (3.29–3.11)
Unique reflections 376,846 753,230 248,179 238,131
Completenessa 99.4 (99.4) 100.0 (99.9) 98.2 (99.4) 95.7 (98.1)
I/σa 15.2 (2.7) 22.7 (7.8) 9.8 (2.3) 8.2 (2.1)
Redundancy 5.2 7.0 3.6 3.7
Rmergea (%) 9.7 (39.1) 7.7 (41.7) 8.6 (35.6) 9.1 (40.1)

Refinement
No. of protein residues 6752 6630 6630
No. of water molecules 4447 229 246
Rworka (%) 20.3 (21.2) 21.2 (29.7) 21.1 (31.0)
Rfreea (%) 23.6 (25.3) 24.2 (34.4) 24.4 (34.1)
Average B-factor (Å2)
Protein 19.2 46.6 45.4
Substrate — 30.0 30.0
Solvent 35.1 39.6 40.5
rmsd from ideal values
Bonds (Å) 0.030 0.022 0.020
Angles (°) 2.27 1.97 1.92
Ramachandran analysis (%)
Most favored 88.5 87.1 84.6
Allowed 11.5 12.9 15.3
a
Numbers in parentheses refer to the highest-resolution shell.

A. Therefore, the GTF-SI structure was compared
with that of Bacillus licheniformis α-amylase (BLA) as
a representative example of GH family 13 amylases,
while the sequence identity between GTF-SI and
BLA is less than 20%. The overall fold of the catalytic
domains A, B and C of GTF-SI was similar to that of
BLA (Fig. 1a).12 Domain A formed the core of the
GTF-SI catalytic domains.5,13 This domain shares a
TIM-barrel motif with GH family 13 α-amylases14
but has two additional helices: the α4′-helix (resi-
dues 592–602) and the α4″-helix (residues 614–627)
(Fig. 1a and Fig. S2). In a previous study, sequence
analysis predicted that the secondary structural
elements of GH family 70 GSases are circularly
permuted as compared to those of GH family 13 α-
amylases,15 and this was confirmed by the current
structure of GTF-SI (Fig. 1a and b). The first helix
(α1) of the GTF-SI TIM-barrel was superimposable
on the third α-helix (α3) of BLA. The two additional
helices, α4′ and α4″, which are not present in the
BLA structure, were positioned between the β4-
strand and the α5-helix of the GTF-SI TIM-barrel
(Fig. 1c and Fig. S2). The observed permutations
extended to domains B and C. Domain B, which
formed a twisted antiparallel sheet of six β-strands,
and domain C, which was composed of eight β-
strands with a Greek key motif (Fig. 1a), are well
conserved among GH family 13 enzymes. 12,16
Domain B, which in BLA is positioned between the
β3-strand and the α3-helix, was composed of two
separate polypeptide chains, one located at the N-
terminus and the other, at the C-terminus of the
TIM-barrel in GTF-SI (Fig. 1b and Fig. S2). Domain
C, which is located at the C-terminal end of the BLA
TIM-barrel, was positioned between the α6-helix
and the β6-strand in GTF-SI. Naturally occurring
circular permutations between homologous proteins
have been reported previously, but cases of such
extensive permutation are very rare.17 As a result of
the structural similarity search by the Dali server,3
domain B of GTF-SI is most similar to that of BLA
[Protein Data Bank (PDB) code 1VJS] with a Z-score
of 2.7. Domain C of GTF-SI is also most similar to that
of the BLA part of chimera enzyme (PDB code 1E3X)
with a Z-score of 9.2. The correlation between these
domains and reaction specificities is not clear because
BLA hydrolyzes the α(1–4) glycosidic linkage. Many
other structures of GH family 70 GSases may be
required to show the role of these domains.18
Domains IV and V are unique to GH family 70
GSases.5 In the GTF-SI structure, domain IV was
positioned next to domain B (Fig. 1a). Domain V is
thought to be composed of sequences upstream of
domain IV1 and/or downstream of domain IV2,
which implies that it is located next to domain IV.
Although domains IV and V are not essential for the
enzymatic activity of GSases,19 domain IV may
serve as a “hinge,” swinging glucan-binding do-
main V toward and away from the catalytic
domains to facilitate glucan extension. The three-
 180
Crystal Structure of Glucansucrase
Fig. 1. Structure of GTF-SI from the dental caries pathogen S. mutans. (a) Ribbon diagrams of GTF-SI and BLA. In GTF-SI, domains IV, A and B are composed of
chains IV1 (orange) and IV2 (salmon), chains A1 (blue) and A2 (purple) and chains B1 (green) and B2 (yellow green), respectively. In BLA, domain B is composed of a
single polypeptide chain, while domain A is composed of two chains, as in GTF-SI. Domains C (magenta) of both enzymes are composed of a single polypeptide chain.
β-Sheets in the TIM-barrels are numbered as they occur in the amino acid sequence. Bound calcium (red) and sodium (black) ions are shown as spheres. (b) Circular
permutation of domains between GTF-SI and BLA. (c) Structure of α-helices α4′ and α4″ of GTF-SI. Acarbose in the active site is shown in a wire frame model. GTF-SI,
BLA and ASase are colored red, green and blue, respectively.
Crystal Structure of Glucansucrase
dimensional structure of domain IV seems to be
unique (Fig. 1a) because no structural homologues
of domain IV were found in the PDB on the Dali
server. The structure of domain V was not resolved
in the current study. A strong electron density (8 σ)
was observed at the interface of domains A and B
(Fig. 1a). This was assigned to a Ca2+ based on its
similarity to the Ca-binding site I of BLA.12
Substrate-binding site structure: Comparison
with amylosucrase
Similar to GSases, Neisseria polysaccharea amylosu-
crase (ASase), which is a GH family 13 amylase, can
synthesize an amylose-like polymer from sucrose.20
GTF-SI synthesizes predominantly α(1–3) glucan
from sucrose, whereas ASases synthesize α(1–4)
glucan from the same substrate. GSases and ASases
are believed to share a similar transglycosylation
mechanism.5,21,22 The glucosyl and fructosyl moieties
of the “primary sucrose” molecule bind to subsite −1
and subsite +1 of the enzyme, respectively. A proton
released from glutamate (Glu515 for GTF-SI) attacks
the glucosidic oxygen. The bound sucrose is then
hydrolyzed, leaving a glycosyl moiety covalently
bound to the aspartic acid residue (Asp477 for GTF-
SI) as an intermediate, which is stabilized by another
aspartic acid residue (Asp588 for GTF-SI). After
fructose is released from subsite +1, the glucosyl
moiety of a “second sucrose” binds to subsite +1. A
glutamate residue (Glu515 for GTF-SI) pulls off the
proton from the hydroxyl group of acceptor sucrose
to produce the activated hydroxyl ion. The hydroxyl
ion engages in nucleophilic attack of the glycosyl-
enzyme intermediate in subsite −1 to form a glucan.
The structure of ASase from N. polysaccharea was
reported by Jensen et al.20 Comparison of the GSase
and ASase structures, despite limited sequence
identity, revealed common components involved
in the transglycosylation in both enzymes. We first
investigated the structure of GTF-SI in complex with
acarbose, which was resolved at 3.1 Å resolution
(Fig. 2a and Fig. S3). Acarbose is a pseudotetrasac-
charide and a strong inhibitor of GSase.8 The Ki of
acarbose for S. mutans GSase is 0.2. Figure 2a shows
the substrate-binding site structure of GTF-SI in
complex with acarbose. The structure of the
complex was identical with the high-resolution
structure of apo GTF-SI (Fig. S4). The clear density
corresponding to acarbose (Fig. S3a and b) allows
unambiguous modeling of the inhibitor. There were
no other binding sites of acarbose in the present
molecule. Figure2a also shows the position of the
glucosyl moiety in the structure of the covalent
glucosyl-enzyme intermediate of the inactive mutant
form of ASase superimposed onto the GSase
structure. The terminal pyranosyl ring of acarbose
in GTF-SI was closely superimposed on the glycosyl
intermediate in subsite − 1 in the ASase structure. By
181
analogy to GH family 13 enzymes, it has been
proposed that three conserved acidic residues of
GTF-SI (Fig. 3), Asp477, Glu515 and Asp588, act as a
nucleophile, a general acid/base catalyst and a
stabilizer of the glucosyl intermediate, respectively,
as described above.5 In addition to these catalytic
residues, Arg475 and His587 (GTF-SI numbering) in
subsite − 1 are well conserved in family 70 enzymes
and family 13 enzymes; Asp909 and Tyr916 are also
conserved between family 70 enzymes and ASase.
The active-site structure among the GH family 13
members is well conserved. Thus, the amino acid
residues such as Arg475, Asp477, Glu515, His587,
Asp588 and Tyr916 constructing subsite − 1 of GTF-
SI are also conserved with those of other GH family
13 amylases, Aspergillus oryzae α-amylase (PDB code
7TAA) and Thermoactinomyces vulgaris R-47 α-amy-
lase 2 (PDB code 3A60). Our results indicated that
recognition of the glucosyl moiety of the primary
sucrose and formation of the glycosyl-enzyme
intermediate at subsite − 1 are well conserved
among GH family 70 and 13 enzymes.
In contrast, residues located in subsite + 1 of GTF-
SI, such as Tyr430, Leu433 and Trp517, are not
conserved in ASase. Interestingly, GSases and ASase
have longer insertion sequences between the β4-
strand and the α5-helix. Overall, the structure of
ASase was comparable to that of GTS-SI; however,
the structure between the β4-strand and the α5-helix
was quite different (Fig. 1c). The insertions are
located adjacent to subsites + 2 and + 3 and seem to
be key factors in transglycosylation specificity.20 In
addition, the subsite + 1 structure of GTF-SI is not
conserved for those of A. oryzae α-amylase (PDB
code 7TAA) and T. vulgaris R-47 α-amylase 2 (PDB
code 3A60) as for ASase. The use of acarbose to
inhibit the GSase activity of S. mutans also results in
the loss of activity of maltase-glucoamylase in the
small intestine, causing hypoglycemia.23 However,
there is no similarity of acarbose recognition
between GTF-SI and maltase-glucoamylase (PDB
code 2QMJ) because the maltase-glucoamylase
belongs to GH family 31.
The Ca-binding site in close proximity to subsite
+ 1 was observed at the interface of domains A and B
(Figs. 1a and 2). This site was composed of the side
chains of Glu431, Asp437, Asp959 and the main-
chain carbonyl oxygen atom of Asn481. The Asn481
side chain formed a hydrogen bond with the C4 and
C6 hydroxyl groups of the acceptor maltose (Fig. 2
and Fig. S3c). Thus, the Ca2+-binding site appeared
to be essential for the formation or stabilization of
subsite + 1.
Transglycosylation specificity: Comparison with
other GSases
To investigate the structural basis of the transgly-
cosylation specificity of GSases in more detail, we
182 Crystal Structure of Glucansucrase

resolved the structure of GTF-SI in complex with structure of apo GTF-SI (Fig. S4) with a clear density
maltose at 3.1 Å resolution (Fig. 2b). The structure of corresponding to maltose (Fig. S5a and b), which
the complex was identical with the high-resolution allowed unambiguous modeling of the molecule.

Fig. 2 (legend on next page)

Crystal Structure of Glucansucrase 183

Fig. 3. Primary structural alignment of three GSases from S. mutans and analysis of sequence diversity using the Pfam
protein family database. Amino acid residues comprising sugar-binding subsites −1, +1 and +2 in GTF-SI are colored red.
The data for these logos consisted of 31 sequences for family 13 enzymes and 12 sequences for family 70 enzymes. The
overall height of the stack indicates the sequence conservation at that position, while the height of the symbols within the
stack indicates the relative frequency of each amino acid at that position. A few sequences with rare insertions were
removed for convenience. Amino acids are colored according to their chemical properties: polar amino acids are green;
basic, blue; acidic, red; and hydrophobic amino acids are black.

There were no other binding sites of maltose in the
present molecule. Maltose is a well-known transgly-
cosyl acceptor for GSases, but not a donor.24 When
maltose serves as an acceptor, the acceptor products
with both GTF-S and GTF-I were panose and a series
of homologues of panose linked to the nonreducing-
end glucosyl unit of maltose by an α(1–6) linkage.24
Indeed, in subsite + 1, the C6 hydroxyl group formed
hydrogen bonds with the carboxyl group of Glu515
(Fig. 2b and Fig. S5c), which has been proposed to

Fig. 2. Substrate-binding site structures of GTF-SI. (a) Stereo view of the substrate-binding site with acarbose. (b) Stereo
view of the substrate-binding site with maltose. (c and d) Proposed sucrose binding modes to clarify the reaction
mechanism of GTF-S, GTF-SI and GTF-I. Acarbose, maltose and sucrose are shown as stick models. Carbon and oxygen
atoms in the models are shown in yellow and red, respectively. A heptacoordinated Ca2+ and water molecules are shown
in sphere models and colored red and cyan, respectively. The glucosyl intermediate in the structure of ASase is
superposed on the GTF-SI structure and is shown in gray. Domains are colored as in Fig. 1a. Green broken lines indicate
the putative interactions between sucrose and Asp593. Computational models of binding sucrose were constructed using
the programs Coot and Molfeat (FiatLux Co., JPN). The central position of glucosyl unit in subsite +1 was fixed to that of
bound maltose.
184
serve as a general acid/base catalyst. Bound
maltose, adopting a low-energy chair–chair confor-
mation, was found in the acceptor-binding site
composed of subsite + 1, subsite + 2 and a part of
subsite + 3 (Fig. S5c). This binding mode of maltose
is inconsistent with the preference of GTF-SI for the
C3 hydroxyl group using the sucrose as substrate.
Fructosyl unit is more bulky than glycosyl unit;
therefore, we presumed that the binding mode of
sucrose is different to that of maltose, as shown in
Fig. 2c and d. Asp593 might interact with the
sucrose directly and might interact with maltose
via water molecules, although water molecules
have not been identified in the ligand-bound
structure of GTF at limited resolution. As de-
scribed later, we concluded that Asp593 is the only
residue that influences the type of glucan products
produced by GTF-S, GTF-SI and GTF-I. In any
case, the proper hydroxyl group was oriented
toward the active center, where the C1 carbon of
the covalent glucosyl-enzyme intermediate in
ASase is located. The glucose moiety in subsite
+ 1 also interacted with Asn481, Tyr430 and
Trp517 (Fig. 2b and Fig. S5c). Trp517 provided
the platform for the acceptor glycosyl moiety, and
Tyr430 participated in hydrophobic interactions with
carbon atoms of the glycosyl moiety in subsite +1.
Asn481, which formed part of the Ca2+-binding site
(discussed above), participated in hydrogen bonding
with the C4 and C6 hydroxyl groups of the glucosyl
moiety in subsite +1. Thus, these residues appeared
to be critical for the recognition of the moiety in
subsite +1.
To further investigate the mechanism of glycosyl
bond formation, we compared the amino acid
sequences of the GH family 70 enzymes (Fig. 3).
GTF-SI and GTF-I are mutansucrases that synthesize
mainly insoluble glucan with α(1–3) glycosidic
linkages. GTF-S is a dextransucrase that synthesizes
predominantly soluble glucan with α(1–6) linkages.5
Most of the amino acid residues located in subsites
+ 1 and + 2 (Fig. 2 and Fig. S2) were conserved in the
three enzymes, with the exception of Glu431 and
Asp593. This suggested that the glycosyl linkage
product is determined by limited differences among
the enzymes. The side chains of Glu431 formed part
of the Ca-binding site, and Glu431 was in a position
opposite the active center. The position of Asp593 in
the α4′-helix appeared to be the most critical point
for acceptor sugar orientation. Shimamura et al.
reported that replacement of Thr at this position in
GTF-S with Asp or Glu promotes the synthesis of
insoluble glucan, likely having more α(1–3) linkages,
while replacement of this Asp residue in GTF-I with
Thr promotes soluble glucan synthesis,25 as the
soluble glucan likely has more α(1–6) linkages.
Moreover, Monchois et al. also reported that
mutation of this position in GTF-I of Leuconostoc
mesenteroides influences the structure and the size of
Crystal Structure of Glucansucrase
the resultant glucan.26 Our results were consistent
with these previous results and suggested that the
distinct reaction specificities of GTF-SI and GTF-S
are due to structural different structures in subsites
+1 and +2, particularly Asp593. In the GTF-SI–
maltose structure, the acceptor sugar was sand-
wiched by Trp517 and Tyr430, and the C6 hydroxyl
group of the glucosyl moiety in subsite +1 was
pointed toward the active center (Fig. 2b). This result
and the preference of GTF-SI for the C3 hydroxyl
group using the sucrose indicate that subsites +1 of
GSases are capable of accepting the glucose in a dual
manner, and a residue, position 593, is the key to
determine the type of glucan products.
The unique structural features of family 70
enzymes in the α4′- and α4″-helices were observed
in the current crystal structure, as described above
(Fig. 1c). The results of random mutagenesis of a
GSase from another oral streptococci, Streptococcus
oralis (47% amino acid identity with GTF-SI)
indicated that the N-terminus of the α4′-helix and
the adjacent loop stabilize the transition state and
determine the specificity of GSases. 27 The N-
terminal end of the α4′-helix and the loop between
the β4-sheet and the α4′-helix could interact with
longer or branched substrates, and modifications in
this region could alter the reaction specificity of
GSases, including regioselectivity and chain length
of the resultant oligosaccharides.25,27
In addition to its role as the glycosyl acceptor,
maltose also is a very weak inhibitor for GSase,
while the effectiveness is about 1 order of magnitude
smaller than that of the acarbose.8 The structure of
GTF-SI in complex with maltose revealed that
maltose serves as a competitive inhibitor of the
second acceptor sucrose (Fig. 2b). This information
should prove to be useful in the design of novel
GSase inhibitors. Acarbose contains a nonhydrolyz-
able nitrogen-linked bond that blocks the catalytic
activity of various GHs, including maltase-glucoa-
mylase in the small intestine.28 This mechanism
could underlie some of the side effects of this
inhibitor, such as hypoglycemia.23 New inhibitors
that specifically target subsites + 1, + 2 and + 3 of
GSase can now be designed based on the structure
of the GTF-SI–maltose complex reported herein.
Most recently, after the submission of this article,
the crystal structure of the enterobacterial GSase
GTF-180, a homologue to GTF-SI, was reported by
Vujicic-Zagar et al.29 While GTF-180 catalyzes both
α(1–6) and α(1–3) glycosidic linkages, the structure
is consistent in principle with that of GTF-SI, a
dental caries pathogenesis factor, which mainly
catalyzes α(1–3) glycosidic linkages.5
Accession numbers
Coordinates and structure factors have been
deposited in the PDB with accession codes 3AIE
Crystal Structure of Glucansucrase
(apo), 3AIC (complexed with acarbose) and 3AIB
(complexed with maltose).
Supplementary materials related to this article can
be found online at .doi:10.1016/j.jmb.2011.02.028
Acknowledgements
This study was supported in part by the
Exploratory Research for Advanced Technology
Iwata Human Receptor Crystallography Project
(Japan Science and Technology Agency) (to S.I.),
the Targeted Proteins Research Program (to S.I. and
T.M.), Grants-in-Aid for Japan Society for the
Promotion of Science Fellows (to K.I.), Grand-in-
Aid for Young Scientists (B) (20780078) (S.I.),
Grants-in-Aid for Scientific Research (B) (to T.K.
and T.S.), a grant from the Research and Develop-
ment Program for New Bioindustry Initiatives (to
K.A.) and Grants-in-Aid for Scientific Research (S)
(to K.A.) in Japan. The X-ray diffraction data
collection was approved by the Photon Factory
Advisory Committee (Proposal 2010G169).
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