Fems Rev v23 p13

Enzymology of one-carbon metabolism in methanogenic pathways
James G. Ferry *
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16801, USA
Received 6 June 1998; accepted 21 September 1998
Abstract
Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding
pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the
two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source
of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway
where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to
coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of
the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the
other major pathway, formate or H
2
is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and
reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines.
In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the
methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide
reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of
understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a
foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed
sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus
jannaschii. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights re-
served.
Keywords: Archaeon; Methanogenesis; One-carbon; Structure and function; Enzymology
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2. Carbon dioxide reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2. Formylmethanofuran dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3. Formylmethanofuran:tetrahydromethanopterin formyltransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4. N
S
,N
IH
-Methenyltetrahydromethanopterin cyclohydrolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5. N
S
,N
IH
-Methylenetetrahydromethanopterin dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
0168-6445 / 99 / $19.00 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 2 9 - 1
FEMSRE 630 8-2-99 Cyaan Magenta Geel Zwart
* Tel. : +1 (814) 863-5721; Fax: +1 (814) 863-6217; E-mail : jgf3@psu.edu
FEMS Microbiology Reviews 23 (1999) 13^38
2.6. N
S
,N
IH
-Methylenetetrahydromethanopterin reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.7. N
S
-Methyltetrahydromethanopterin:coenzyme M methyltransfrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.8. Methyl-coenzyme M reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3. Fermentation of acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2. Acetate kinase and phosphotransacetylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.3. CO dehydrogenase/acetyl-CoA synthase complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.4. Carbonic anhydrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4. Disproportionation of methanol and methylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.2. Methanol : coenzyme M methyltransferase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.3. Monomethylamine:coenzyme M methyltransferase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.4. Dimethylamine:coenzyme M methyltransferase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.5. Trimethylamine:coenzyme M methyltransferase system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1. Introduction
A signicant fraction of the earth's biosphere
contains oxygen-free environments where anaerobic
microbes convert complex organic matter to methane
and carbon dioxide constituting an integral compo-
nent of the global carbon cycle. They reside in vast
and diverse habitats such as the rumen, the lower
intestinal tract, sewage digesters, landlls, freshwater
sediments of lakes and rivers, rice paddies, hydro-
thermal vents, coastal marine sediments and the
subsurface [137]. The process of converting complex
organic matter to simple one-carbon compounds,
representing the most oxidized (CO
2
) and reduced
(CH
4
) forms of carbon, requires a consortium of at
least three interacting metabolic groups of anae-
robes. The rst two groups are primarily from the
Bacteria domain and convert the organic matter to
H
2
, CO
2
, formate, and acetate. The third group, the
methanoarchaea, are members of the Archaea do-
main and convert the metabolic products of the rst
two groups to methane. One-carbon reactions play
important roles in the metabolism of all three
metabolic groups and are dominant in the metha-
noarchaea. The past two decades have witnessed
elucidation of one-carbon pathways for methan-
ogenesis revealing unusual biochemical reactions
requiring novel enzymes and cofactors (Fig. 1).
More recent biochemical studies have provided a
rm foundation for a new era of discovery
focused on the structure/function of these novel
enzymes.
Several reviews have appeared recently which em-
phasize either the general metabolism [8,35] or bio-
energetics [23] of the methanoarchaea. This review
emphasizes developments within the past ve years
concerning the enzymology of one-carbon reactions
in methanogenic pathways with the view to prepare
the reader for the exciting discoveries beginning to
emerge from investigations into the structure/func-
tion of these novel enzymes.
2. Carbon dioxide reduction
2.1. Background
The reduction of CO
2
to CH
4
is accomplished
with electrons derived from the oxidation of either
H
2
or formate (Eqs. 1 and 2).
4H
2
CO
2
3CH
4
2H
2
O 1
4HCOO
3
4H
3CH
4
8CO
2
2H
2
O 2
Eq. 1 is the sum of Eq. 3a, Eq. 4a, Eqs. 5^7, Eq.
8a, Eq. 8b and Eqs. 9^12. Eq. 2 is the sum of Eq. 3b,
Eq. 4b, Eqs. 5^7, Eq. 8a and Eqs. 9^12. This section
focuses on enzymes catalyzing one-carbon reactions
(Eqs. 5^7, Eq. 8a, Eq. 8b and Eqs. 9^11). The reader
is directed to the following references which discuss
J.G. Ferry / FEMS Microbiology Reviews 23 (1999) 13^38 14
enzymes catalyzing Eq. 3a, Eq. 3b, Eq. 4a, Eqs. 4b
and 12 [23,34,99,113].
2H
2
34o
3
4H
3a
2HCOOH32CO
2
4o
3
4H
3b
2H
2
2F
420
32F
420
H
2
4a
2HCOOH 2F
420
32F
420
H
2
2CO
2
4b
CO
2
MF 2o
3
2H
3foinyl3MF H
2
O 5
foinyl3MF H
4
MI133foinyl3H
4
MI1 MF
6
3foinyl3H
4
MI1 H
3
Y 103nolhonyl3H
4
MI1
H
2
O 7
Y 103nolhonyl3H
4
MI1
F
420
H
2
3Y 103nolhylono3H
4
MI1 F
420
H
8a
Y 103nolhonyl3H
4
MI1
H
2
3Y 103nolhylono3H
4
MI1 H
8b
Y 103nolhylono3H
4
MI1
F
420
H
2
33nolhyl3H
4
MI1 F
420
9
3nolhyl3H
4
MI1 HS3CoM3CH
8
3S3CoM
H
4
MI1 10
CH
8
3S3CoM HS3CoL3CH
4
CoM3S3S3CoL
11
CoM3S3S3CoL 2o
3
2H
3HS3CoM
HS3CoL 12
2.2. Formylmethanofuran dehydrogenase
The enzyme functions reversibly to reduce CO
2
to
the formyl level producing formylmethanofuran (Eq.
5). The vGP for this reaction is +16 kJ mol
31
[23]
and at low H
2
partial pressures encountered in the
environment is even more positive; thus, it is pro-
posed that either a sodium gradient driven by Eq. 10
or a proton gradient driven by Eq. 12 drives Eq. 5.
The structure of methanofuran (MF) is shown in
Fig. 1. The electron donor for the dehydrogenase is
unknown [59]. The enzyme from Methanosarcina
barkeri contains ve distinct subunits and contains
molybdopterin guanine dinucleotide (Fig. 1), 30 non-
heme iron and 30 acid-labile sulde [60,61]. The en-
zyme utilizes CO
2
rather than HCO
3
8
as the active
species and catalyzes a MF-dependent exchange be-
tween CO
2
and the formyl group of formyl-MF con-
sistent with N-carboxymethanofuran as an inter-
mediate in the reaction [129]. The genes encoding
the ve subunits form a transcription unit (fmdE-
FACDB) and are cotranscribed with a gene, fmdF,
predicted to encode a polyferredoxin possibly con-
taining eight [4Fe-4S] clusters [130]. FmdB has de-
duced sequence similarity to the formate dehydro-
genase of Methanobacterium formicicum which also
contains a molybdopterin cofactor [58] suggesting
FmdB is the catalytic subunit. In addition, FmdB
has the potential to bind one [4Fe-4S] cluster. The
polyferredoxin could potentially donate electrons di-
rectly to FmdB. M. thermoautotrophicum contains
both molybdenum and tungsten isoforms of formyl-
methanofuran dehydrogenase [49]. The tungsten en-
zyme harbors a `molybdopterin'
1
dinucleotide which
can contain either molybdenum or tungsten depend-
ing on the growth conditions, a property contrasting
with other tungsten enzymes in nature which contain
tungsten in `molybdopterin' mononucleotide [50].
The M. thermoautotrophicum tungstoenzyme is com-
posed of four subunits (FwdABCD), the genes for
which are cotranscribed with three other genes
(fwdHFGDACB) [50]. Sequence comparisons suggest
FwdB binds the molybdopterin dinucleotide and that
FwdF is a polyferredoxin and FwdG is a clostridial
type ferredoxin. The deduced sequence of FwdH
contains two motifs that could bind [4Fe-4S] clus-
1
The term `molybdopterin' implies a molybdenum-containing
cofactor; however, the term is also used for the cofactors that
contain tungsten and can be misleading.
C
Fig. 2. A: Ribbon diagram of the formyltransferase tetramer illustrating the extended contact between subunits 1 and 2 (red and yellow),
and subunits 3 and 4 (blue and green). B: Ribbon diagram of the monomer. The L strands are in red, K helices in green, and loops in
yellow.
Fig. 1. Structures of cofactors required for catalysis of one-carbon reactions in methanogenic pathways.
ters. The M. thermoautotrophicum molybdoenzyme
contains three subunits (FmdABC) [49] encoded on
a transcriptional unit (fmdECB). Sequence compari-
sons show FmdB has high identity with FwdB and
FmdB from M. barkeri indicating all three are mo-
lybdopterin binding subunits. The FmdA subunit
has the same apparent molecular mass and N-termi-
nal sequence as FwdA; furthermore, Southern blot-
ting indicates only one DNA sequence encoding the
N-terminal sequence leading to the proposal that the
tungsten and molybdenum enzymes share this sub-
unit which is also consistent with the genomic se-
quence [112]. Using heterologous oligonucleotide
probes for fwdB from M. thermoautotrophicum and
fmdB from M. barkeri, two genes were isolated from
Methanopyrus kandleri with deduced sequences
which suggested this hyperthermophile contains
two tungsten isozymes of formylmethanofuran dehy-
drogenase, one of which is a novel selenoenzyme
[131]. The results also suggest that M. kandleri
does not have a molybdoenzyme consistent with
the exclusive use of tungsten in enzymes from hyper-
thermophilic microbes; likewise, the genomic
sequence of the hyperthermophile M. jannaschii con-
tains only genes encoding a tungsten formylmetha-
nofuran dehydrogenase [12].
2.3. Formylmethanofuran:tetrahydromethanopterin
formyltransferase
This enzyme catalyzes the transfer of the formyl
group from formyl-MF to H
4
MPT (Eq. 6). The
structure of H
4
MPT is shown in Fig. 1. The formyl-
transferase has been puried and characterized from
mesophilic (M. barkeri), thermophilic (M. thermo-
autotrophicum), and hyperthermophilic (M. kandleri
and Methanothermus fervidus) methanoarchaea [73].
The puried enzymes contain one type of subunit
with a molecular mass of approximately 32 kDa
and exhibit a sequential kinetic mechanism consis-
tent with formation of a ternary complex. The crys-
tal structure of the enzyme from M. kandleri,
determined to 1.73 A
resolution, indicates a homo-

tetramer more accurately described as a dimer of
dimers [31] (Fig. 2A). The subunit contains two
tightly associated lobes (Fig. 2B), each of which
Fig. 3. Active sites of coenzyme F
420
coenzyme F
420
H
2
, N
,N
10
-methenyl-H
4
MPT
, and N
,N
10
-methylene-H
4
MPT. Complete F
420
and
H
4
MPT structures are shown in Fig. 1.
has a core fold consisting of an antiparallel four-
stranded L sheet anked by two K helices identifying
the formyltransferase as a member of the large K+L
structure family of proteins. The similarity in the
basic architecture of both lobes suggests a gene du-
plication event has contributed to evolution of the
enzyme. The surface of the protein contains an un-
usually high number of negative charged residues
which is proposed to account for the tolerance to
high salt. Unfortunately, structures complexed with
either substrate were not obtained which precluded
the accurate identication of an active site or pro-
posal of a mechanism.
The ftr mRNA from M. barkeri and M. fervidus
[73,76] is monocistronic and does not form an oper-
on as has been suggested for the ftr from M. ther-
moautotrophicum [25]. The genes encoding each of
the four formyltransferases studied have a surprising
degree of sequence identity (minimally 46%) when
considering the phylogenetic and physiological diver-
sity of these methanoarchaea. The genomic sequence
of M. thermoautotrophicum [112] predicts two ftr
genes; however, the genomic sequence of M. janna-
schii [12] predicts only one suggesting that only one
formyltransferase is essential. There is no signicant
sequence similarity to any known protein in the da-
tabases; however, an aerobic methylotroph from the
Bacteria domain (Methylobacterium extorquens
AM1) contains an open reading frame encoding a
MF- and H
4
MPT-dependent formyltransferase with
a deduced sequence showing strong identity to for-
myltransferases from the methanoarchaea [17]. M.
extorquens also contains a methenyl-H
4
MPT cyclo-
hydrolase encoded by an open reading frame with a
deduced sequence strongly identical to cyclohydro-
lases from the methanoarchaea. These results raise
interesting questions regarding the origin, evolution,
and function of enzymes involved in one carbon me-
tabolism. For example, did the homologous genes
evolve from a common ancestor, or were they trans-
ferred horizontally? Genomic sequencing has re-
vealed a large number of fundamental reactions in
the Archaea and Bacteria domains that are catalyzed
by similar enzymes having high identity between do-
mains arguing that the universal ancestor was highly
developed [12,72,95,112] ; however, analysis of more
sequences are necessary before the question of the
origin of these genes can be properly resolved.
2.4. N
S
,N
IH
-Methenyltetrahydromethanopterin
cyclohydrolase
The methenyl-H
4
MPT cyclohydrolase catalyzes
Eq. 7. The enzyme from M. thermoautotrophicum
[24,91], M. kandleri [11], and M. barkeri [118] con-
tains two identical subunits of 37^41 kDa and has no
identiable prosthetic groups. The gene encoding the
enzyme from M. thermoautotrophicum is apparently
transcribed monocistronically. Comparison of the
deduced sequence with six other tetrahydrometha-
nopterin-dependent enzymes reveal no sequence sim-
ilarities which excludes identication of a consensus
tetrahydromethanopterin binding site. Heterologous
production of the active enzyme portends a crystal
structure and proposed mechanism [125].
2.5. N
S
,N
IH
-Methylenetetrahydromethanopterin
dehydrogenases
Two genetically distinct dehydrogenases have been
described, one coenzyme F
420
-dependent (catalyzing
Eq. 8a) and another H
2
-dependent (catalyzing Eq.
8b). Coenzyme F
420
(Fig. 1) is an obligate two-
electron carrier (redox midpoint potential near
3350 mV) that donates or accepts a hydride ion.
The F
420
-dependent enzyme has been puried from
M. barkeri, [29,120], M. thermoautotrophicum
[91,121], and M. kandleri [69]. All are composed of
one type of subunit (30^36 kDa), as either a hexamer
or an octamer, with no detectable prosthetic group.
The catalytic mechanism is ternary, a result consis-
tent with a direct hydride transfer to or from F
420
.
The hydride transfer has Re-face specicity at C14a
of methylenetetrahydromethanopterin and Si-face
specicity at the C5 of F
420
(Fig. 3) [71]. The Re-
face specicity suggests methylene-H
4
MPT adopts a
conformation where the Re hydrogen-carbon bond is
antiperiplanar to the two lone pair orbitals on N
and N
10
of H
4
MPT when bound to the enzyme.
The dehydrogenase joins ve other F
420
-dependent
enzymes from the methanoarchaea in having
Si-face-only specicity. This result is particularly in-
teresting since pyridine nucleotides are functionally
similar to F
420
but pyridine nucleotide-dependent en-
zymes exhibit either Si-face or Re-face specicity.
The catalytic mechanism and structural basis for
Si-face-only stereospecicity of F
420
-dependent en-
zymes are likely forthcoming now that the mtd genes
have been cloned and heterologous production of the
functional enzymes from M. thermoautotrophicum
[92] and M. kandleri [70] has been reported. The
mtd genes from these two methanoarchaea are tran-
scribed monocistronically. A comparison of these
two mtd sequences with the putative mtd gene deter-
mined from genomic sequencing of Methanococcus
jannaschii [12] indicates greater than 50% identity
among the three. Sequence analysis of mtd from
M. thermoautotrophicum suggests a potential F
420
binding site in the N-terminus [92].
Characterization of the H
2
-dependent dehydrogen-
ases from M. thermoautotrophicum strain Marburg
[140], M. kandleri [86], Methanobacterium wolfei
[141], and Methanococcus thermolithotrophicus [46]
indicates the enzymes contain one type of subunit
(38^42 kDa) and have no detectable prosthetic
groups or metals except zinc [141]. The enzymes
are not inhibited by metal binding compounds, are
unable to catalyze an H
2
/H
exchange in the absence

of substrate and cannot reduce dyes with H
2
, char-
acteristics which are hallmarks of classical hydrogen-
ases. Furthermore, the sequence deduced from the
hmd genes have no metal binding motifs character-
istic of classical hydrogenases [46,94,128,141] ; yet,
the enzyme is sensitive to O
2
. What then could the
mechanism be for this clever anomaly of nature? The
catalytic mechanism has been investigated by meas-
uring the distribution of H
2
, HD, and D
2
produced
from CH
2
NH
4
MPT (methylene-H
4
MPT) in D
2
O or
from CD
2
NH
4
MPT in H
2
O by mass spectrometry
[107], and the formation of
18
CH
2
NH
4
MPT and
18
CHDNH
4
MPT from
18
CHOH
4
MPT
(methenyl-
H
4
MPT
) and H
2
or D
2
in H
2
O or D
2
O by NMR
[106]. The results of these studies indicate that the
dehydrogenase catalyzes the stereospecic transfer of
an hydride ion from H
2
into the pro-R position of
the methylene carbon. The enzyme also catalyzes a
stereospecic exchange of the pro-R hydrogen with
water leading to the proposal that CHOH
4
MPT
reduction with H
2
involves a pentacoordinated car-
bonium ion (CH
8
NH
4
MPT) transition state inter-
mediate where the pro-R hydrogen bond is proto-
nated and exchanges with water (Fig. 4) [67,68].
In fed-batch cultures where H
2
is abundant, tran-
scription of hmd (encoding the H
2
-dependent dehy-
drogenase) is favored over transcription of mtd (en-
coding the F
420
-dependent dehydrogenase) whereas
the inverse is observed under culture conditions
where H
2
is limiting [89,94]. Assays reveal dierences
in relative activities of the two dehydrogenases in
H
2
-limiting vs. H
2
-nonlimiting chemostat cultures
that would be expected from the transcription results
[127]. Under culture conditions where expression of
Fig. 5. Proposed reduction of F
420
with H
2
catalyzed by the
metal-free methylenetetrahydromethanopterin dehydrogenase sys-
tem. CH
2
NH
4
MPT, methylenetetrahydromethanopterin; CHO
H
4
MPT
, methenyltetrahydromethanopterin. Hmd, H
2
-depend-
ent methylenetetrahydromethanopterin dehydrogenase; Mtd,
F
420
-dependent methylenetetrahydromethanopterin dehydrogen-
ase.
Fig. 4. The reaction catalyzed by the H
2
-dependent methylenetetrahydromethanopterin dehydrogenase. The structure in brackets is the
proposed pentacoordinated C14a carbocation intermediate. CH
2
NH
4
MPT, methylenetetrahydromethanopterin; CHOH
4
MPT
, methenyl-
tetrahydromethanopterin. The complete H
4
MPT structure is shown in Fig. 1.
the F
420
-dependent dehydrogenase is favored, the ox-
idation of H
2
is accomplished by the nickel-contain-
ing F
420
-dependent hydrogenase (Eq. 4a) which has a
ten-fold higher anity for H
2
(K
n
=0.02 mM) com-
pared to the H
2
-dependent dehydrogenase (K
n
=0.2
mM) ([2]). These results have led to the conclusion
that under H
2
-nonlimiting conditions the H
2
-de-
pendent dehydrogenase substitutes for the F
420
-de-
pendent dehydrogenase catalyzing the reduction of
CHOH
4
MPT
to CH
2
NH
4
MPT. However, the
physiological functions of the F
420
- and H
2
-depend-
ent dehydrogenases may be more complex. In nickel
limited chemostat cultures of M. thermoautotrophi-
cum the activity of nickel-containing F
420
-dependent
hydrogenase (Eq. 4a) is undetectable whereas activ-
ities of the F
420
-dependent and H
2
-dependent dehy-
drogenases are four- and six-fold higher [2]. These
results suggest that under nickel limitation the two
metal-free dehydrogenases function in tandem to ox-
idize H
2
and reduce F
420
(Fig. 5) thereby replacing
the nickel-containing F
420
-dependent hydrogenase
(Eq. 4a). The genomic sequences of M. thermoauto-
trophicum [112] and M. jannaschii [12] predict two
additional putative hmd genes demanding further ex-
periments to determine the specic physiological
functions of the gene products.
2.6. N
S
,N
IH
-Methylenetetrahydromethanopterin
reductase
The reductase catalyzes Eq. 9. The enzymes puri-
ed from M. thermoautotrophicum [84,85,119], M.
kandleri [83], and M. barkeri [87,120] are F
420
-de-
pendent, contain one subunit (35^38 kDa) with no
discernable prosthetic groups, and exhibit a ternary
complex kinetic mechanism suggesting direct hydride
transfer. The genes (mer) encoding the reductases
from M. thermoautotrophicum strains Marburg and
vH are transcribed monocistronically [93,126]. Com-
parison of the deduced sequence with databases re-
vealed signicant similarity (25^30%) with F
420
- and
avin-dependent enzymes in species from the Bacte-
ria domain; however, no consensus F
420
binding mo-
tif was identied. Curiously, signicant similarity
was not found with any of the published sequences
for F
420
-dependent enzymes from microbes in the
Archaea domain. These results again raise questions
regarding the origin and evolution of enzymes from
the Bacteria and Archaea domains which have com-
mon functions.
2.7. N
S
-Methyltetrahydromethanopterin:coenzyme M
methyltransfrase
The methyltransferase catalyzing Eq. 10 is an in-
tegral membrane-bound complex which requires so-
dium ions for activity and, in addition to methyl
transfer, functions to generate a sodium ion gradient
across the membrane [5]. The enzyme characterized
from M. thermoautotrophicum [36,37,62] and Meth-
anosarcina strain Go 1 [78,80], contains a corrinoid
cofactor (5P-hydroxybenzimidazolyl cobamide, Fig.
1) of which the Co
1
atom functions as a super-re-
duced nucleophile accepting the methyl group from
CH
8
-H
4
MPT in the rst of two partial reactions cat-
alyzed by the enzyme. The second partial reaction
involves transfer of the methyl group from CH
8
-
Co
8
to coenzyme M (HS-CoM, Fig. 1) producing
CH
8
-S-CoM and regenerating the activated Co
1
form of the corrinoid. The methyl transfer step is
Fig. 6. Predicted membrane orientation of the MtrA subunit of
the methyltetrahydromethanopterin:coenzyme-M methyltransfer-
ase from M. thermoautotrophicum. His denotes the histidine resi-
due proposed to form the lower axial ligand to the cobalt atom
of the methylated corrinoid.
dependent on sodium and presumably functions to
pump this cation across the membrane [37,136]. The
methyltransferase complex from M. thermoautotro-
phicum contains eight non-identical subunits (34,
28, 24, 23, 21, 13, 12.5, and 12 kDa) (MtrA-H) for
which the encoding genes have been cloned and se-
quenced [45,117]. The genes form an operon
(mtrEDCBAFGH) located between the methyl-coen-
zyme M reductase I operon (mcr) and a downstream
open reading frame predicted to encode a Na
/Ca
,
K
exchanger [45], the latter consistent with a pro-

posed function for the methyltransferase in the gen-
eration of a sodium gradient. The deduced sequences
of MtrB, MtrC, MtrD, and MtrA suggest they are
integral membrane proteins. MtrA contains corri-
noid [36] and, from the deduced sequence and im-
munolabeling, is thought to be only partially inte-
grated into the cytoplasmic face of the cell
membrane with the corrinoid binding site protruding
into the cytoplasm [117]. MtrA has been heterolo-
gously produced in Escherichia coli as a soluble trun-
cated apoprotein minus 25 hydrophobic C-terminal
residues proposed to anchor the protein to the mem-
brane [43]. Denaturation and refolding of the protein
in the presence of cobalamin produced a corrinoid-
containing holoprotein. Electron paramagnetic reso-
nance (EPR) spectroscopy of the holoprotein dier-
entially labeled with
14
N (nuclear spin 1) and
1
N
(nuclear spin 1/2) reveals a base-o form of the
bound corrinoid with the nitrogen atom of histidine
serving as the lower axial ligand to the Co
2
atom.
Based on the assumption that histidine is also a low-
er axial ligand to Co
8
but not Co
1
, it is hypothe-
sized that sodium ion translocation is coupled to a
conformational change in MtrA when Co
1
is me-
thylated and histidine binds to CH
8
-Co
8
(Fig. 6).
Sequence alignment of the four known MtrA se-
quences identies a consensus corrinoid binding mo-
tif containing a conserved histidine that is a candi-
date for the proposed ligand to CH
8
-Co
8
[79]. The
recent cloning, sequencing, and expression of genes
encoding the methyltransferase from M. mazei shows
that all eight subunits are heterologously produced
in E. coli laying the foundation for future studies to
determine the function of the other seven subunits
[79].
Fig. 7. A: Molecular surface representation of methyl-CoM reductase illustrating the entrance (white arrow) of one of the two channels.
Subunits K and KP are red and orange, L and LP in dark green and light green, and Q and QP in dark blue and light blue. B: Same as in A
except with KP removed exposing the interior of the two channels (entrances marked by white arrows) and the cofactor binding sites. F
480
is in yellow and the heterodisulde CoM-S-S-CoB is in white.
2.8. Methyl-coenzyme M reductase
The reductase, catalyzing Eq. 11, is common to all
methanogenic pathways. The enzymes from Metha-
nosarcina mazei [22,122], Methanosarcina thermophila
[53], Methanosaeta soehngenii [57], and M. kandleri
[101] have been investigated; however, the enzymes
from M. thermoautotrophicum have received the
most attention. The electron donor for all reductases
is coenzyme B (CoB) (Fig. 1) and the heterodisulde
CoM-S-S-CoB is a product in addition to CH
4
. M.
thermoautotrophicum contains two genetically dis-
tinct isozymes (MCRI and MCRII) both of which
have native molecular masses of approximately 300
kDa and are composed of three dierent subunits
with molecular masses of 65 (K), 46 (L), and 30^35
(Q) kDa in an K
2
L
2
Q
2
conguration [102]. The native
isozymes each contain two molecules of coenzyme
F
480
(F
480
), a yellow nickel-containing porphinoid
(Fig. 1), that is at the active site of the enzyme.
The isozymes from M. thermoautotrophicum have
the same EPR spectroscopic properties and ternary-
complex kinetic mechanism; however, the pH opti-
ma for activity and kinetic constants are signicantly
dierent [10]. MCR I has a K
nCoL
of 0.1^0.3 mM, a
K
nnolhylSCoM
of 0.6^0.8 mM, and a V
nax
of ap-
proximately 6 Wmol min
31
mg
31
. On the other hand,
MCR II has a K
nCoL
of 0.4^0.6 mM, a
K
nnolhylSCoM
of 1.3^1.5 mM, and a V
nax
of ap-
proximately 21 Wmol min
31
mg
31
. The pH optimum
of MCR I is 7.0^7.5 and for MCR II is 7.5^8.0.
The genes encoding several reductases from phy-
logenetically diverse methanoarchaea (mcrBGA) are
cotranscribed in operons (mcrBDCGA) with two ad-
ditional genes [4,9,18,66,134,135]. The MCRII-en-
coding operons from M. thermoautotrophicum [98]
and Methanothermus fervidus [77] are similar except
they are missing the gene equivalent to mcrC.
Although expressed in M. thermoautotrophicum [28]
and Methanococcus vannielii [108,115,116], there is
no known function for McrD or McrC. Transcrip-
tion of the operons encoding the two isozymes of M.
thermoautotrophicum is growth phase-dependent with
the MCRII enzyme only transcribed early in batch
cultures and replaced by MCRI later in the growth
phase [94,98]. This dierential regulation has been
correlated to the supply of H
2
where MCRI is ex-
pressed at low H
2
concentrations and MCRII at rel-
atively higher concentrations of H
2
[89,127] analo-
gous to the dierential expression of the F
420
- and
H
2
-dependent dehydrogenases; however, H
2
may
not be the only controlling factor as medium reduc-
tant also appears to play a role [96]. Factor F
800
, a
degradation product of F
420
produced in cells under
oxidative stress [47,124], is proposed to play a role in
the regulation of both the methylreductase and the
F
420
- and H
2
-dependent dehydrogenases [97].
The recent crystal structure of the MCRI isozyme
from M. thermoautotrophicum has shed light on the
active site and mechanism [30]. The two F
480
cofac-
tors are positioned at the bottom of identical narrow
channels which are formed by residues from the
KKPLQ or KPKLPQP subunits (Fig. 7). The F
480
mole-
cules are separated by approximately 50 A
which
precludes a requirement for both and indicates two
independent catalytic sites. High resolution struc-
tures, complexed with either HS-CoM plus HS-
CoB or CoM-S-S-CoB, lead to a proposed reaction
mechanism (Fig. 8) largely consistent with previous
proposals [7,55]. The relative positions of CoM,
CoB, and F
480
in the crystal structure is consistent
with a nucleophilic attack of Ni(I) on CH
8
-S-CoM
and formation of a [F
480
]Ni(III)-CH
8
intermediate
(step 1, Fig. 8). The role of Ni(I) in catalysis was
recently supported by reductive activation of the in-
active oxidized enzyme when F
480
is reduced to the
Ni(I) redox state by titanium(III)citrate [6,38]. In the
next step (step 2, Fig. 8), the Ni(III) oxidizes HS-
CoM producing
c
S-CoM thiyl radical and
[F
480
]Ni(II)-CH
8
intermediates. In step 3 (Fig. 8),
protonolysis releases CH
4
and the thiyl radical is
coupled to
3
S-CoB to form CoB-S-S-CoM with
the excess electron transferred to Ni(II) forming
Ni(I). Evidence for the proposed carbon-nickel
bond will be necessary to conrm this hypothesis.
3. Fermentation of acetate
3.1. Background
Most of the methane produced in nature derives
from the methyl group of acetate (Eq. 13).
CH
8
CO
3
2
H
2
O3CH
4
HCO
3
8
13
Fig. 8. Representation of the proposed steps in the mechanism of methyl-CoM reductase. See text.
Eq. 13 is the sum of Eqs. 14^20 which dene the
pathway for M. thermophila.
CH
8
COO
3
A1I3CH
8
CO
2
IO
23
8
ADI 14
CH
8
CO
2
IO
23
8
HS3CoA3CH
8
COSCoA Ii
15
CH
8
COSCoA H
4
SI1 H
2
O3CH
8
3H
4
SI1
2o
3
2H
CO
2
HS3CoA 16
CH
8
3H
4
SI1 HS3CoM3CH
8
3SCoM H
4
SI1
17
CO
2
H
2
O3HCO
3
8
H
18
CH
8
3S3CoM HS3CoL3CH
4
CoM3S3S3CoL 19
Ii ADI CoM3S3S3CoL 2o
3
8H
3HS3CoM HS3CoL A1I H

2
O 20
The carbon-carbon bond of acetate is cleaved fol-
lowed by reduction of the methyl group to methane
with electrons originating from oxidation of the
carbonyl group to carbon dioxide; thus, the pathway
is a true fermentation. The pathway is the same in
methanosaeta species except acetate thiokinase
(CH
8
COO
3
+CoA+ATPCCH
8
COSCoA+AMP+
PPi) replaces Eqs. 14 and 15. The reactions involving
carbon ow that are unique to the acetate fermenta-
tion pathway are Eqs. 14^16 and 18. All others are
similar to those described in the carbon dioxide re-
duction pathway.
3.2. Acetate kinase and phosphotransacetylase
Together, these enzymes activate acetate to acetyl-
CoA (Eqs. 14 and 15) prior to cleavage by the CO
dehydrogenase/acetyl-CoA synthase complex (Eq.
16). The genes encoding acetate kinase and phospho-
transacetylase from M. thermophila have been cloned
and sequenced [74], and shown to be co-transcribed
on an operon [110]. Both enzymes have been hyper-
produced in E. coli in an highly active form [74]
which has allowed site-specic replacement of resi-
dues to probe the catalytic sites.
Acetate kinase puried from M. thermophila is an
K
2
homodimer with a subunit molecular mass of
45 kDa. A mechanism has been proposed in which
an unspecied glutamate is phosphorylated to form a
covalent phosphoryl-enzyme intermediate during cat-
alysis. Alteration of E384 in the M. thermophila en-
zyme results in either undetectable or extremely low
kinase activity suggesting this glutamate is essential
for catalysis and consistent with the proposed mech-
anism [111]. Alteration of the neighboring E385 in-
uenced the K
n
values for acetate and ATP with
only moderate decreases in k
cal
which suggests in-
volvement in substrate binding but not catalysis.
The unaltered enzyme is not inactivated by N-ethyl-
maleimide; however, replacement of E385 with cys-
teine confers sensitivity towards the inhibitor which
can be prevented by preincubation with substrates
conrming a location for E385 in the active site.
Crystals of the acetate kinase from M. thermophila
diracting to below 1.7 A
are reported which sug-

gests a structure is imminent [16].
The phosphotransacetylase from M. thermophila is
a monomeric enzyme with a molecular mass of
35 kDa. Based on inhibition by N-ethylmaleimide,
a ternary mechanism was proposed for phospho-
transacetylases in which an unspecied cysteine ab-
stracts a proton from CoASH forming a nucleophilic
thiolate anion attacking acetyl phosphate [48]. Re-
placement of all four cysteines in the M. thermophila
enzyme with alanine shows that only C159 is essen-
tial for activity and, therefore, is a candidate for
involvement in catalysis [100]. Activity of the unal-
tered phosphotransacetylase was sensitive to N-ethyl-
maleimide. Inhibition kinetics of the cysteine var-
iants indicates the sensitivity results from
modication of C312 which is at the active site but
itself is nonessential for catalysis.
3.3. CO dehydrogenase/acetyl-CoA synthase complex
The ve-subunit (K, L, Q, N, O) CO dehydrogenase/
acetyl-CoA synthase (CODH/ACS) complex is cen-
tral to the pathway and functions to cleave the C-C
and C-S bonds in the acetyl moiety of acetyl-CoA,
oxidize the carbonyl group to CO
2
(CO dehydrogen-
ase activity), and transfer the methyl group to
H
4
SPT (Eq. 16). Tetrahydrosarcinapterin (H
4
SPT) is
an analog of H
4
MPT [123]. The synthase complex
contains three enzyme components. The nickel/iron-
sulfur (Ni/Fe-S) component contains the K (CdhA)
and O (CdhB) subunits. The corrinoid/iron-sulfur
(Co/Fe-S) component contains the N (CdhD) and Q
(CdhE) subunits of the complex [1]. The third com-
ponent, the L (CdhC) subunit, is unstable and has
not been characterized. There are three metal clusters
(A, B, and C) in the Ni/Fe-S component [81] which
have EPR spectroscopic properties indistinguishable
from clusters A, B, and C in the K
2
L
2
CODH/ACS
from the acetogenic anaerobe Clostridium thermoace-
ticum. Cluster A is the proposed site for synthesis or
cleavage of the C-C and C-S bonds of acetyl-CoA
and is a novel Ni-X-[Fe
4
S
4
] metal center where X is
an unknown bridging atom [99]. Cluster C is the
proposed site for CO dehydrogenase activity and
also a novel bimetallic Ni-X-[Fe
4
S
4
] cluster [51].
Cluster B is a conventional Fe
4
S
4
center thought to
shuttle electrons to and from cluster C. Based on
similarity of the EPR spectrum with the clostridial
enzyme, it is proposed that cluster A in the Ni/Fe-S
component of the M. thermophila CODH/ACS com-
plex is the site for cleavage of acetyl-CoA with trans-
fer of the methyl group to the Co/Fe-S component
[54,81].
The cdh genes encoding the ve subunits of the
CODH/ACS complex form an operon [88], the tran-
scription of which is regulated in response to the
growth substrate [114]. An additional open reading
frame is cotranscribed with the genes encoding the
ve subunits suggesting the gene product may be
required for maturation of any of the ve subunits
of the complex; indeed, the deduced sequence of this
open reading frame has high identity to CooC which
is required for insertion of nickel into the CO dehy-
drogenase of Rhodospirillum rubrum [64]. CdhB con-
tains only one histidine and no cysteines suggesting
metal clusters A, B, and C of the Ni/Fe-S component
are localized to CdhA. The sequence of CdhA con-
tains several cysteine and histidine motifs which are
perfectly conserved with the CdhA sequence from
the acetotrophic methanoarchaeon M. soehngenii,
and therefore are candidates for coordination of
metal clusters A, B, and C [88]. The K (AcsA) sub-
unit of the clostridial CODH/ACS contains cluster A
[138] ; however, there is no signicant identity be-
tween it and CdhA from M. thermophila suggesting
convergent evolution to cluster A. On the other
hand, CdhC is highly identical to the C-terminal
half (residues 317^729) of the clostridial K (AcsA)
subunit which contains cluster A. Both sequences
contain perfectly conserved four- and two-cysteine
motifs that are almost certain to coordinate cluster
A (Fig. 9). There are no other cysteine residues in
CdhC from M. thermophila strongly suggesting only
one metal cluster, cluster A, in this subunit. The
function of this proposed cluster A in CdhC is un-
known; however, a proteolytic fragment of CdhC
from the M. barkeri CODH/ACS complex has ace-
tyl-CoA/CoA exchange activity which at least im-
plies C-S bond cleavage of acetyl-CoA [40].
The M. thermophila CdhA sequence has two fer-
redoxin-like CXXCXXCXXXCP motifs which could
coordinate two B clusters [88], although it can not be
ruled out that only one cluster B is present in CdhA.
CdhA also has signicant identity (22 and 26%) to
the sequences deduced from the genes encoding the
CO dehydrogenase from R. rubrum [63] and the L
subunit of the CODH/ACS from C. thermoaceticum
[90], both of which contain cluster C but not cluster
A [51]. Several regions are conserved among these
proteins including a cysteine (CX
2
CX
4
CX
0
CG) and
Fig. 9. Alignment of the predicted sequences of the CdhC subunit from the CODH/ACS of M. thermophila (Mt CdhC) and K subunit
from C. thermoaceticum (Ct AcsA). Identical (:) and functionally similar (.) amino acid residues are noted. Conserved cysteine residues
are highlighted and underlined.
a histidine (HX
2
HX
2
H) motif which may be in-
volved in coordination of metal cluster C.
The CdhE subunit of the Co/Fe-S component of
the M. thermophila CODH/ACS complex is highly
identical with the sequence deduced from the gene
encoding the L subunit of the analogous corrinoid/
iron-sulfur protein from C. thermoaceticum [88]. The
sequence CXXCXXXXCX
16
CP in the N-terminus is
perfectly conserved which suggests involvement in
chelation of the Fe
4
S
4
centers identied by EPR
spectroscopy [54]. The two subunits of the Co/Fe-S
component from M. thermophila have been inde-
pendently produced in E. coli. The CdhE subunit
binds corrinoid and contains an iron-sulfur center
consistent with the deduced sequence. The bound
corrinoid is methylated with CH
8
-H
4
SPT (unpub-
lished results) suggesting CdhE accepts the methyl
group from the Ni/Fe-S component and then do-
nates it to H
4
SPT.
The proposed mechanism for acetyl-CoA cleavage
by the CODH/ACS complex from M. thermophila
(Fig. 10) is consistent with the biochemical and bio-
physical studies and the deduced sequences of the
subunits, all of which support a reversal of the mech-
anism proposed for synthesis of acetyl-CoA by the
well characterized clostridial system [99]. In the pro-
posed mechanism, acetyl-CoA binds to the CdhC
subunit where the C-S bond is cleaved and the acetyl
group is transferred to the nickel atom of cluster A
in the CdhA subunit of the Ni/Fe-S component (step
1, Fig. 10). In step 2, C-C bond cleavage takes place
at cluster A and the methyl group is transferred to
the corrinoid prosthetic group of the CdhE subunit
from the Co/Fe-S component (step 3). In the nal
step, the carbonyl group is released from cluster A of
CdhA as CO. The Ni/Fe-S component has CO de-
hydrogenase activity and, therefore, it is proposed
that the CO migrates to cluster C of CdhA where
it is oxidized to CO
2
[81].
M. barkeri also contains a ve-subunit CODH/
ACS enzyme complex with biochemical properties
indistinguishable from the complex in M. thermophi-
la [39^42]. Acetate-grown M. soehngenii synthesizes a
CODH/ACS complex containing a K
2
L
2
component
which is analogous to the Ni/Fe-S component from
M. thermophila [56]. Although the K
2
L
2
component
from M. soehngenii has CO dehydrogenase and ace-
tyl-CoA cleavage activities suggesting the presence of
metal cluster A, EPR spectroscopy identies only
clusters B and C. A high-spin signal ascribed to a
Fe
6
S
6
prismane cluster is detected in the M. soehn-
genii enzyme; however, the function of this proposed
metal center is unknown. Recently, an enzyme with
CO dehydrogenase activity from acetate-grown
Methanosarcina frisia was described which has a sub-
unit composition and EPR spectral properties similar
to the K
2
L
2
component from the M. soehngenii
CODH/ACS complex [27]. The signicance of the
prismane-like cluster and apparent lack of cluster
A in the M. soehngenii and M. frisia enzymes are
important questions, the resolution of which will
likely shed light on the reaction mechanism.
The genomic sequences of M. thermoautotrophi-
cum and M. jannaschii contain genes homologous
Fig. 10. Proposed mechanism of acetyl-CoA cleavage by the CODH/ACS complex involving subunits CdhA and CdhE. X, unidentied
bridging ligand.
to cdhA, cdhC, cdhD, and cdhE [12,112]. Both of
these methanoarchaea employ the CO
2
-reduction
pathway and are unable to ferment acetate; how-
ever, they are autotrophic and able to synthesize all
cellular components starting with acetyl-CoA synthe-
sized by CODH/ACS. The reduction of CO
2
to
methyl-H
4
MPT supplies the methyl group of ace-
tyl-CoA and the carbonyl group originates from
the reduction of CO
2
to CO [109]. It has been hy-
pothesized that the universal ancestor was autotro-
phic based on the presence of key enzymes involved
in carbon xation in the Bacteria and Archaea do-
mains that share high sequence identity between do-
mains [95]. If autotrophy was a very early invention,
it is possible that the CODH/ACS was rst used to
synthesize acetyl-CoA and later evolved for utilizing
acetate as an energy source. On the other hand, evi-
dence has been presented that acetate may have been
produced on prebiotic earth [52] presenting the pos-
sibility that CODH/ACS was invented rst to cleave
acetate as a source of energy and later evolved to
synthesize acetyl-CoA for autotrophic growth as ace-
tate was depleted.
3.4. Carbonic anhydrase
This enzyme catalyzes hydration of CO
2
to car-
bonic acid (Eq. 18). The carbonic anhydrase (Cam)
puried from acetate-grown M. thermophila is a ho-
motrimer with a subunit molecular mass of 23 kDa
[3,65]. The deduced sequence of the gene (cam) en-
coding Cam has no signicant identity with the se-
quence of any known carbonic anhydrases described
from the K and L classes which are comprised mostly
of enzymes from mammalian and plant sources;
therefore, Cam is the prototype of a new class (the
Q class) of carbonic anhydrase. A search of the data-
bases reveals Cam has signicant identity with sev-
eral open reading frames of unknown function in
diverse microbes. The identity extends to the histi-
dine motif ligating the active site zinc in Cam sug-
gesting Q-class carbonic anhydrases are distributed
Fig. 11. A: Side view ribbon diagram of the Cam monomer. The L strands are shown as curved arrows in purple and K helices as rib-
bons in cyan. The active site Zn
2
ion is shown with the van der Waals surface in yellow. B: View of the trimer along the 3-fold axis.
across the Bacteria and Archaea domains. These re-
sults also suggest carbonic anhydrases are more wide
spread in prokaryotes than previously thought and
may provide diverse functions in microbial metabo-
lism.
The cam gene encodes an additional 34 N-terminal
residues not present in the puried enzyme [3]. These
residues have properties characteristic of signal pep-
tides in secretory proteins suggesting Cam may be
located outside the cell membrane. This proposed
location could possibly facilitate the ecient removal
of cytoplasmic CO
2
by conversion to HCO
3
8
outside
the cell membrane. The energy yield for the metha-
nogenic fermentation of acetate is low under stand-
ard conditions (vGP =336 kJ mol
31
, Eq. 13); thus,
the ecient removal of cytoplasmic CO
2
could im-
prove the thermodynamics of the pathway.
The crystal structure of Cam heterologously pro-
duced in E. coli reveals a novel left-handed parallel
L-helix fold (Fig. 11A) with no similarity to any
known carbonic anhydrases [65]. This fold is of par-
ticular interest since it contains only left-handed
crossover connections between the parallel L-strands,
which are a rare occurrence. The three active-site
zinc ions are each located at the interface between
two monomers (Fig. 11B). Each zinc is ligated with
three histidines, two from one subunit and the third
from an adjacent subunit. Apart from the histidines
ligating zinc, the active-site residues of Cam are sig-
nicantly dierent from the human carbonic anhy-
drases (K class) (Fig. 12). In the human enzyme,
Glu106 and Thr199 form an H bond network with
zinc-bound hydroxyl orienting the lone pair of elec-
trons for attack on CO
2
. Based on the orientations
of Glu62 and Gln75 in the crystal structure (Fig. 12),
it is proposed that these residues function in analogy
to Glu106 and Thr199 of the human enzyme.
4. Disproportionation of methanol and methylamines
4.1. Background
The conversion of methanol and methylamines to
methane and carbon dioxide (Eq. 23) is a dispropor-
tionation event in which the methyl group of one
substrate molecule is oxidized to CO
2
(Eq. 21) pro-
viding six electrons for reduction of the methyl
Fig. 12. Superposition of the active site zinc ions and coordinating His residues of the Cam and human carbonic anhydrases. Short
stretches of C trace are shown as thick and thin lines for Cam and the human enzyme, respectively. Side chains shown in ball and stick
representation and labeled in black (Cam) or green (human) are: (I) zinc-bound water, (ii) the side chains of residues involved in zinc co-
ordination, and (iii) residues either known or proposed to be involved in catalysis.
groups of three substrate molecules to methane (Eq.
22).
I3CH
8
2H
2
O3IH CO
2
6o
3
6H
21
8I3CH
8
6o
3
6H
38CH
4
8IH 22
4I3CH
8
2H
2
O34IH 8CH
4
CO
2
23
(where R=-SH, -OH, -NH
2
, -NHCH
8
, -N(CH
8
)
2
, or
-NCH
8
8
).
Eq. 21 is accomplished by transfer of the methyl
group to H
4
SPT and oxidation to CO
2
by a reversal
of Eqs. 5^7, Eqs. 8a and 9 in the CO
2
reduction
pathway. Methyl transfer to H
4
SPT has not been
investigated; however, methyl group reduction is
known to begin with transfer to CoM catalyzed by
methyltransferase systems specic for each substrate.
The nal step involves reduction to methane cata-
lyzed by the methyl-CoM reductase common to all
methanogenic pathways. This section describes the
methyltransferase systems which are unique to the
pathways for disproportionation of methanol and
methylamines.
4.2. Methanol : coenzyme M methyltransferase
system
The enzyme system from M. barkeri contains two
components which catalyze sequential reactions
(Eqs. 24 and 25) leading to an overall transfer of
the methyl group of methanol to HS-CoM (Eq. 26).
CH
8
OH Co
1
MlaLC
H
3Co
8
3CH
8
MlaLC H
2
O 24
HSCoM Co
8
3CH
8
MlaLC3Co
1
MlaLC
CH
8
3SCoM H
25
CH
8
OH HSCoM3CH
8
3SCoM H
2
O 26
The methanol :corrinoid methyltransferase (MT1)
component contains the subunits MtaB (50 kDa)
plus MtaC (27 kDa) and autocatalyzes methylation
of its corrinoid (Eq. 24) tightly bound to MtaC [103].
The methyl-corrinoid:HSCoM methyltransferase
(MT2-M) component catalyzes Eq. 25 and contains
only one 36-kDa subunit (MtaA) with no prosthetic
groups. The methanol :corrinoid methyltransferase
has been puried from M. barkeri with the cobalt
atom of the corrinoid in the inactive Co
2
redox
state that can be reactivated by reduction to Co
1
with titanium(III)citrate [103], a result consistent
with a mechanism in which Co
1
acts as a super
nucleophile attacking activated methanol. The genes
encoding the subunits are transcribed as a mtaCB
unit [103]. The deduced sequence of the corrinoid-
containing MtaC is 35% similar to the cobalamin
binding domain of E. coli methionine synthase [26]
where the cofactor is bound in the base-o congu-
ration with a histidine serving as the lower axial
ligand. The 11-amino acid motif containing the his-
tidine of methionine synthase is strictly conserved in
MtaC suggesting the corrinoid is also base-o and
could potentially be ligated to this conserved histi-
dine in the lower axial position. This prediction has
been conrmed by EPR spectroscopy and site di-
rected replacement studies of heterologously pro-
duced MtaC [104]. MtaB, independently produced
in E. coli, catalyzes the methylation of free cobala-
min in the Co
1
redox state (vGP =37 kJ mol
31
)
indicating this subunit activates methanol for nucle-
ophilic attack by MtaC [105]. MtaB contains one
zinc and activity is dependent on zinc or cobalt pre-
senting the possibility these metals function as Lewis
acids in the activation of methanol.
The gene encoding MtaA (catalyzing Eq. 25) is
transcribed monocistronically and is in higher levels
in methanol-grown cells [44], consistent with the pro-
posed function [33]. MtaA has been produced in an
active form in E. coli [44,75]. MtaA (MT2-M) also
contains zinc [75] and methylation of HSCoM with
methylcobalamin is dependent on this metal [105] ;
thus, it is postulated that zinc binds to and activates
HSCoM for nucleophilic attack on the methyl group
of methylcobalamin. Based on these results, a mech-
anism is proposed for the methyltransferase system
in which MtaB activates methanol for nucleophilic
attack by the corrinoid of MtaC which then transfers
the methyl group to HSCoM that has been activated
by MtrA [105].
During in vivo and in vitro turnover, the active
Co
1
form of corrinoid-containing enzymes is occa-
sionally oxidized which inactivates the enzymes ne-
cessitating a mechanism for reduction to the active
redox state of cobalt [82]. For example, reactivation
of E. coli methionine synthase is achieved by reduc-
tive methylation of the cobalt where adenosylmethio-
nine and NADPH are the methyl and electron do-
nors. In the case of MT1 (MtaB/MtaC), a chemical
reducing system comprised of titanium(III)citrate is
utilized in vitro [103]. In vivo reactivation of MT1 is
achieved with H
2
, hydrogenase, ferredoxin, ATP,
and methyltransferase activation protein (MAP)
[19]. MAP, a monomer of 60 kDa, is autophos-
phorylated by ATP and does not contain prosthetic
groups [21]. A three-step mechanism is proposed
[20]. In step 1, the cobalt atom of the corrinoid is
in the Co
8
redox state and is reduced to Co
2
with
ferredoxin, hydrogenase and H
2
which releases water
as the upper axial ligand to cobalt. In step 2, MAP-
phosphate converts the corrinoid from the base-on
conguration (coordinated with N-3 of 5-hydroxy-
benzimidazole as the lower axial ligand to cobalt)
to base-o. In non-protein bound corrinoids, the
midpoint potential of the Co
1
/Co
2
redox couple
for the base-on form (E
o
P =V592 mV) is nearly
100 mV more negative than for base-o; thus, it is
postulated that conversion to the base-o form by
MAP-phosphate raises the midpoint potential of the
Co
1
/Co
2
redox couple allowing for reduction to
Co
1
at the low H
2
concentrations present in the
cell environment. It is further hypothesized that the
binding of methanol assists MAP-phosphate in the
reduction to Co
1
. This hypothesis is in analogy to
the proposed binding of ATP and methyl-H
4
MPT to
the methyl-H
4
MPT:HS-CoM methyltransferase in a
ternary complex thereby raising the midpoint poten-
tial of the Co
1
/Co
2
redox couple by 200 mV to the
level of physiological electron donors [80].
4.3. Monomethylamine:coenzyme M
methyltransferase system
The monomethylamine:coenzyme M (MMA:
CoM) methyltransferase system from M. barkeri
contains two catalytic components [14]. The rst
component contains monomethylamine methyltrans-
ferase (MMAMT) paired with monomethylamine
corrinoid protein (MMCP). MMAMT is a 170-kDa
enzyme containing 52-kDa subunits and no pros-
thetic groups [14]. MMAMT transfers the methyl
group of monomethylamine to the corrinoid pros-
thetic group of the 29-kDa MMCP [13]. Thus
MMAMT and MMCP are analogous to MtaB and
MtaC in the methanol :coenzyme M methyltransfer-
ase system and are likely to have similar properties.
The MMA:CoM methyltransferase assay was per-
formed in the presence of titanium(III)citrate; thus,
it is unknown if an enzymatic reductive activation
system is present for specic reduction of the cobalt
atom of MMCP to Co
1
. The genes encoding
MMCP and MMAMT (mtmC and mtmB) form an
operon (mtmCB) [15]. The deduced sequence of
MtmC contains corrinoid binding motifs similar to
the methionine synthase of E. coli including the his-
tidyl residue ligating the cobalt atom of the corri-
noid. The second component of the MMA:CoM
methyltransferase system is methyl-MMCP:CoM
methyltransferase (MT2-A), an isozyme of MT2-M
[139]. The 36-kDa MT2-A also contains zinc [75] and
the encoding gene (designated cmtA in [75], and
mtbA in [44]) has a deduced sequence which is 37%
identical to the deduced sequence for the MT2-M
gene (designated cmtM in [75], and mtaA in [44])
which includes the proposed zinc binding domain.
The mtbA gene is transcribed monocistronically [44].
4.4. Dimethylamine:coenzyme M
methyltransferase system
A requirement for MT2-A in crude extracts was
established for the dimethylamine:coenzyme M
(DMA:CoM) methyltransferase system in M. barkeri
[33,132] suggesting a two-component system; indeed,
a corrinoid-containing protein fraction which sup-
ports only DMA:CoM but not TMA:CoM methyl-
transferase activity was reported [132]. Recently, a
dimethylamine:5-hydroxybenzimidazolylcobamide
methyltransferase (DMA-MT) was puried from M.
barkeri that together with partially puried MT2-A
catalyzed the stoichiometric conversion of dimethyl-
amine (apparent K
n
=0.45 mM) and HS-CoM to
monomethylamine and methyl-SCoM [133]. The ho-
modimeric DMA-MT has a native molecular mass of
100 kDa and contains one corrinoid. DMA-MT dif-
fers from the rst component of the MMA:CoM
and TMA:CoM (see below) methyltransferase two-
component systems in that the rst component in the
latter two systems are heterodimers with only one of
the subunits containing a corrinoid. The as-isolated
DMA-MT is inactive, but is reductively reactivated
with MAP, ATP and dimethylamine resulting in
methylation of the corrinoid prosthetic group.
4.5. Trimethylamine:coenzyme M methyltransferase
system
The trimethylamine (TMA):CoM methyltransfer-
ase system from M. barkeri also contains two com-
ponents [32], the rst of which is a colorless 52-kDa
protein called TMA-52 that is associated with a cor-
rinoid-containing 26-kDa polypeptide termed TCP
(TMA corrinoid protein). The second component is
MT2-A; however, MT2-M can also substitute albeit
with lower activity. A role for TMA-52 has not been
established; however, it is hypothesized that this pro-
tein acts to methylate TCP with TMA prior to CoM
methylation by MT2 in analogy to the methanol :
CoM and MMA:CoM methyltransferase systems.
Activity of the TMA:CoM system reconstituted
with puried components is dependent on titaniu-
m(III)citrate consistent with a requirement for reduc-
tion of the cobalt atom of TCP to the Co
1
redox
state. The titanium(III)citrate replaced a requirement
for ATP in crude extracts suggesting an ATP-de-
pendent activating system is operable in vitro for
the TMA:CoM methyltransferase. Indeed, evidence
has been presented that MAP is a component in the
activating systems for MMA:CoM, DMA:CoM,
and TMA:CoM methyltransferases [132].
The TMA:CoM methyltransferase system cata-
lyzes methylation of CoM with TMA, but not dime-
thylamine (DMA) or MMA, and the only product is
DMA. The MMA:CoM methyltransferase system
does not utilize either TMA or DMA. Both methyl-
transferase systems share the same MT2 component
[32,132] ; however, the N-termini for TMA-52 and
MMAMT share no signicant identity suggesting
the specicity for substrates lies in these two proteins
and their associated corrinoid proteins TCP and
MMCP.
5. Conclusions
Research within the past ve years has revealed
novel enzymes catalyzing one-carbon reactions in
several methanogenic pathways. Recent research
has also included the cloning and sequencing of
genes encoding these enzymes providing new insights
regarding physiology, enzyme evolution, regulation
of gene expression, and hypotheses for mechanisms
of catalysis. Many of these enzymes have been over-
produced in E. coli opening the way for crystal struc-
tures and site-directed mutagenesis to investigate
structure-function relationships and enzyme mecha-
nism. These already exciting prospects will surely be
eclipsed by exploitation of the recently completed
sequences of the genomes for the methanoarchaea
M. thermoautotrophicum and M. jannaschii ; less
than half of the predicted protein coding regions
can be assigned a role from database sequences.
Acknowledgments
Research at the Pennsylvania State University was
supported by Grants DE-FG02-95ER20198 from the
Department of Energy, and GM44661-07 from the
National Institutes of Health.
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Enzymology of one-carbon metabolism in methanogenic pathways

resolution, indicates a homo-

exchange in the absence

exchanger [45], the latter consistent with a pro-

3HS3CoM HS3CoL A1I H

are reported which sug-

X-ray structure of B12-binding domains of methionine syn-

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