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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-yieldingpathways 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 HP 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. NS,NIH-Methenyltetrahydromethanopterin cyclohydrolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.5. NS,NIH-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

* Tel.: +1 (814) 863-5721; Fax: +1 (814) 863-6217; E-mail: [email protected]

FEMS Microbiology Reviews 23 (1999) 13^38

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2.6. NS,NIH-Methylenetetrahydromethanopterin reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.7. NS-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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294.1. 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 (COP) and reduced

(CHR) 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

HP, COP, 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 COP to CHR is accomplished

with electrons derived from the oxidation of either

HP or formate (Eqs. 1 and 2).

RrP gyP3grR PrPy 1

Rrgyy3 Rr3grR QgyP PrPy 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

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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.


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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 subunithas 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 containstwo 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 HRMPT (Eq. 6). The

structure of HRMPT 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 FRPH coenzyme FRPHHP, NS,NIH-methenyl-HRMPT

, and NS,NIH-methylene-HRMPT. Complete FRPH and

HRMPT structures are shown in Fig. 1.


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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 residueswhich 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 HRMPT-dependent formyltransferase with

a deduced sequence showing strong identity to for-myltransferases from the methanoarchaea [17]. M.

extorquens also contains a methenyl-HRMPT 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 inthe 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. NS,NIH-Methenyltetrahydromethanopterin

cyclohydrolase

The methenyl-HRMPT 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 theenzyme 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. NS,NIH-Methylenetetrahydromethanopterin

dehydrogenases

Two genetically distinct dehydrogenases have been

described, one coenzyme FRPH-dependent (catalyzing

Eq. 8a) and another HP-dependent (catalyzing Eq.

8b). Coenzyme FRPH (Fig. 1) is an obligate two-

electron carrier (redox midpoint potential near

3350 mV) that donates or accepts a hydride ion.

The FRPH-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 FRPH.

The hydride transfer has Re-face specicity at C14a

of methylenetetrahydromethanopterin and Si-face

specicity at the C5 of FRPH (Fig. 3) [71]. The Re-

face specicity suggests methylene-HRMPT adopts a

conformation where the Re hydrogen-carbon bond is

antiperiplanar to the two lone pair orbitals on NS

and NIH of HRMPT when bound to the enzyme.

The dehydrogenase joins ve other FRPH-dependent

enzymes from the methanoarchaea in havingSi-face-only specicity. This result is particularly in-

teresting since pyridine nucleotides are functionally

similar to FRPH 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 FRPH-dependent en-


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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 FRPHbinding site in the N-terminus [92].

Characterization of the HP-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 HP/H exchange in the absence

of substrate and cannot reduce dyes with HP, 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 OP. What then could the

mechanism be for this clever anomaly of nature? Thecatalytic mechanism has been investigated by meas-

uring the distribution of HP, HD, and DP produced

from CHPNHRMPT (methylene-HRMPT) in DPO or

from CDPNHRMPT in HPO by mass spectrometry

[107], and the formation of IQCHPNHRMPT andIQCHDNHRMPT from

IQCHOHRMPT (methenyl-

HRMPT) and HP or DP in HPO or DPO by NMR

[106]. The results of these studies indicate that the

dehydrogenase catalyzes the stereospecic transfer of

an hydride ion from HP 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 CHOHRMPT

reduction with HP involves a pentacoordinated car-

bonium ion (grQNHRMPT) 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 HP is abundant, tran-

scription of hmd (encoding the HP-dependent dehy-

drogenase) is favored over transcription of mtd (en-

coding the FRPH-dependent dehydrogenase) whereas

the inverse is observed under culture conditions

where HP is limiting [89,94]. Assays reveal dierencesin relative activities of the two dehydrogenases in

HP-limiting vs. HP-nonlimiting chemostat cultures

that would be expected from the transcription results

[127]. Under culture conditions where expression of

Fig. 5. Proposed reduction of FRPH with HP catalyzed by the

metal-free methylenetetrahydromethanopterin dehydrogenase sys-

tem. CHPNHRMPT, methylenetetrahydromethanopterin; CHO

HRMPT, methenyltetrahydromethanopterin. Hmd, HP-depend-

ent methylenetetrahydromethanopterin dehydrogenase; Mtd,

FRPH-dependent methylenetetrahydromethanopterin dehydrogen-

ase.

Fig. 4. The reaction catalyzed by the HP-dependent methylenetetrahydromethanopterin dehydrogenase. The structure in brackets is the

proposed pentacoordinated C14a carbocation intermediate. CHPNHRMPT, methylenetetrahydromethanopterin; CHOHRMPT, methenyl-

tetrahydromethanopterin. The complete HRMPT structure is shown in Fig. 1.


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the FRPH-dependent dehydrogenase is favored, the ox-

idation of HP is accomplished by the nickel-contain-

ing FRPH-dependent hydrogenase (Eq. 4a) which has a

ten-fold higher anity for HP (Km = 0.02 mM) com-

pared to the HP-dependent dehydrogenase (Km =0.2

mM) ([2]). These results have led to the conclusion

that under HP-nonlimiting conditions the HP-de-

pendent dehydrogenase substitutes for the FRPH-de-pendent dehydrogenase catalyzing the reduction of

CHOHRMPT to CHPNHRMPT. However, the

physiological functions of the FRPH- and HP-depend-

ent dehydrogenases may be more complex. In nickel

limited chemostat cultures of M. thermoautotrophi-

cum the activity of nickel-containing FRPH-dependent

hydrogenase (Eq. 4a) is undetectable whereas activ-

ities of the FRPH-dependent and HP-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 HP and reduce FRPH (Fig. 5) thereby replacing

the nickel-containing FRPH-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. NS,NIH-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 FRPH-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 FRPH- and

avin-dependent enzymes in species from the Bacte-ria domain; however, no consensus FRPH binding mo-

tif was identied. Curiously, signicant similarity

was not found with any of the published sequences

for FRPH-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. NS-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 CoI atom functions as a super-re-

duced nucleophile accepting the methyl group fromCHQ-HRMPT in the rst of two partial reactions cat-

alyzed by the enzyme. The second partial reaction

involves transfer of the methyl group from CHQ-

CoQ to coenzyme M (HS-CoM, Fig. 1) producing

CHQ-S-CoM and regenerating the activated CoI

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.


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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 colias 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 IRN (nuclear spin 1) and ISN

(nuclear spin 1/2) reveals a base-o form of thebound corrinoid with the nitrogen atom of histidine

serving as the lower axial ligand to the CoP atom.

Based on the assumption that histidine is also a low-

er axial ligand to CoQ but not CoI, it is hypothe-

sized that sodium ion translocation is coupled to a

conformational change in MtrA when CoI is me-

thylated and histidine binds to CHQ-CoQ (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 CHQ-CoQ

[79]. Therecent 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 RQHis in yellow and the heterodisulde CoM-S-S-CoB is in white.

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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 themost 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 CHR. 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 KPLPQP conguration [102]. The native

isozymes each contain two molecules of coenzyme

FRQH (FRQH), 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 Kmgof of 0.1^0.3 mM, a

Kmmtlgow of 0.6^0.8 mM, and a Vm of ap-

proximately 6 Wmol min3I mg3I. On the other hand,

MCR II has a Kmgof of 0.4^0.6 mM, a

Kmmtlgow of 1.3^1.5 mM, and a Vm of ap-

proximately 21 Wmol min3I mg3I. 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 HP where MCRI is ex-

pressed at low HP concentrations and MCRII at rel-

atively higher concentrations of HP [89,127] analo-

gous to the dierential expression of the FRPH- and

HP-dependent dehydrogenases; however, HP may

not be the only controlling factor as medium reduc-

tant also appears to play a role [96]. Factor FQWH, a

degradation product of FRPH produced in cells under

oxidative stress [47,124], is proposed to play a role inthe regulation of both the methylreductase and the

FRPH- and HP-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 FRQH 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 FRQH 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 FRQH in the crystal structure is consistent

with a nucleophilic attack of Ni(I) on CHQ-S-CoM

and formation of a [FRQH]Ni(III)-CHQ 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 FRQH 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 cS-CoM thiyl radical and

[FRQH]Ni(II)-CHQ intermediates. In step 3 (Fig. 8),

protonolysis releases CHR and the thiyl radical is

coupled to 3S-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).

grQgy3

P rPy3grR rgy3

Q 13


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Fig. 8. Representation of the proposed steps in the mechanism of methyl-CoM reductase. See text.


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Eq. 13 is the sum of Eqs. 14^20 which dene the

pathway for M. thermophila.

grQgyy3 e3grQgyPy

P3Q eh 14

grQgyPyP3Q r3goe3grQgygoe i

15

grQgygoe rR rPy3grQ3rR

P3 Pr gyP r3goe 16

grQ3rR r3gow3grQ3gow rR

17

gyP rPy3rgy3

Q r 18

grQ33gow r3gof3grR

gow3

3

3

gof 19

i eh gow333gof P3 Qr

3r3gow r3gof e rPy 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(CHQCOO

3+CoA+ATPCCHQCOSCoA+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 COdehydrogenase/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

KP 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 Km values for acetate and ATP with

only moderate decreases in kt 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. thermophilaenzyme 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 COP (CO dehydrogen-

ase activity), and transfer the methyl group to


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HRSPT (Eq. 16). Tetrahydrosarcinapterin (HRSPT) is

an analog of HRMPT [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 KPLP 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-[FeRSR] 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-[FeRSR] cluster [51].

Cluster B is a conventional FeRSR center thought toshuttle 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 readingframe 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 onlyone 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%) tothe 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 (CXPCXRCXWCG) 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.

J.G. Ferry / FEMS Microbiology Reviews 23 (1999) 13^3826

8/6/2019 Fems Rev v23 p13

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a histidine (HXPHXPH) 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 CXXCXXXXCXITCP in the N-terminus is

perfectly conserved which suggests involvement in

chelation of the FeRSR 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 boundcorrinoid is methylated with CHQ-HRSPT (unpub-

lished results) suggesting CdhE accepts the methyl

group from the Ni/Fe-S component and then do-

nates it to HRSPT.

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 COP [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. soehngeniisynthesizes a

CODH/ACS complex containing a KPLP component

which is analogous to the Ni/Fe-S component from

M. thermophila [56]. Although the KPLP 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

FeTST 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 KPLP component from the M. soehngenii

CODH/ACS complex [27]. The signicance of theprismane-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.

J.G. Ferry / FEMS Microbiology Reviews 23 (1999) 13^38 27

8/6/2019 Fems Rev v23 p13

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to cdhA, cdhC, cdhD, and cdhE [12,112]. Both of

these methanoarchaea employ the COP-reduction

pathway and are unable to ferment acetate; how-

ever, they are autotrophic and able to synthesize allcellular components starting with acetyl-CoA synthe-

sized by CODH/ACS. The reduction of COP to

methyl-HRMPT supplies the methyl group of ace-

tyl-CoA and the carbonyl group originates from

the reduction of COP 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 tosynthesize 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 COP 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 (theQ 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 ZnP ion is shown with the van der Waals surface in yellow. B: View of the trimer along the 3-fold axis.


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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]. Theseresidues 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 COP by conversion to rgy3

Q outside

the cell membrane. The energy yield for the metha-

nogenic fermentation of acetate is low under stand-

ard conditions (vGP =336 kJ mol3I, Eq. 13); thus,

the ecient removal of cytoplasmic COP 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 COP. 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 COP (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.

J.G. Ferry / FEMS Microbiology Reviews 23 (1999) 13^38 29

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groups of three substrate molecules to methane (Eq.

22).

3grQ PrPy3r gyP T3 Tr 21

Q3grQ T3 Tr3QgrR Qr 22

R3grQ PrPy3Rr QgrR gyP 23

(where R = -SH, -OH, -NHP, -NHCHQ, -N(CHQ)P, or

-xgrQQ ).

Eq. 21 is accomplished by transfer of the methyl

group to HRSPT and oxidation to COP by a reversal

of Eqs. 5^7, Eqs. 8a and 9 in the COP reduction

pathway. Methyl transfer to HRSPT 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 ofthe methyl group of methanol to HS-CoM (Eq. 26).

grQyr goIwtfg

r3goQ3grQwtfg rPy 24

rgow goQ3grQwtfg3goIwtfg

grQ3gow r 25

grQyr rgow3grQ3gow rPy 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 CoP redox

state that can be reactivated by reduction to CoI

with titanium(III)citrate [103], a result consistent

with a mechanism in which CoI

acts as a supernucleophile 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 CoI redox state (vGP =37 kJ mol3I)

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) istranscribed 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 nucleophilicattack 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

CoI form of corrinoid-containing enzymes is occa-

sionally oxidized which inactivates the enzymes ne-


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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 isachieved with HP, 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 CoQ redox state and is reduced to CoP with

ferredoxin, hydrogenase and HP 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 CoI/CoP redox couple

for the base-on form (EoP =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

CoI/CoP redox couple allowing for reduction to

CoI at the low HP concentrations present in the

cell environment. It is further hypothesized that the

binding of methanol assists MAP-phosphate in the

reduction to CoI. This hypothesis is in analogy tothe proposed binding of ATP and methyl-HRMPT to

the methyl-HRMPT:HS-CoM methyltransferase in a

ternary complex thereby raising the midpoint poten-

tial of the CoI/CoP 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. barkericontains 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 activationsystem is present for specic reduction of the cobalt

atom of MMCP to CoI. 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 Km = 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


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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 CoI 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 investigatestructure-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.

References

[1] Abbanat, D.R. and Ferry, J.G. (1991) Resolution of compo-

nent proteins in an enzyme complex from Methanosarcina

thermophila catalyzing the synthesis or cleavage of acetyl-

CoA. Proc. Natl. Acad. Sci. USA 88, 3272^3276.[2] Afting, C., Hochheimer, A. and Thauer, R.K. (1998) Function

of HP-forming methylenetetrahydromethanopterin dehydro-

genase from Methanobacterium thermoautotrophicum in coen-

zyme FRPH reduction with HP. Arch. Microbiol. 169, 206^

210.

[3] Alber, B.E. and Ferry, J.G. (1994) A carbonic anhydrase from

the archaeon Methanosarcina thermophila. Proc. Natl. Acad

Sci. USA 91, 6909^6913.

[4] Allmansberger, R., Bokranz, M., Krockel, L., Schallenberg, J.

and Klein, A. (1989) Conserved gene structures and expres-

sion signals in methanogenic archaebacteria. Can. J. Micro-

biol. 35, 52^57.

[5] Becher, B., Muller, V. and Gottschalk, G. (1992) NS-Methyl-

tetrahydromethanopterin:coenzyme M methyltransferase ofMethanosarcina strain Go1 is an Na-translocating membrane

protein. J. Bacteriol. 174, 7656^7660.

[6] Becker, D.F. and Ragsdale, S.W. (1998) Activation of methyl-

SCoM reductase to high specic activity after treatment of

whole cells with sodium sulde. Biochemistry 37, 2639^

2647.

[7] Berkessel, A. (1991) Methyl-coenzyme M reductase: model


8/6/2019 Fems Rev v23 p13

21/26

studies on pentadentate nickel complexes and a hypothetical

mechanism. Bioorg. Chem. 19, 101^115.

[8] Blaut, M. (1994) Metabolism of methanogens. Antonie van

Leeuwenhoek 66, 187^208.

[9] Bokranz, M. and Klein, A. (1987) Nucleotide sequence of the

methyl coenzyme M reductase gene cluster from Methanosar-

cina barkeri. Nucleic Acids Res. 15, 4350^4351.

[10] Bonacker, L.G., Baudner, S., Morschel, E., Bocher, R. and

Thauer, R.K. (1993) Properties of the two isoenzymes of

methyl-coenzyme-M reductase in Methanobacterium ther-

moautotrophicum. Eur. J. Biochem. 217, 587^595.

[11] Breitung, J., Schmitz, R.A., Stetter, K.O. and Thauer, R.K.

(1991) NS,NIH-Methenyltetrahydromethanopterin cyclohydro-

lase from the extreme thermophile Methanopyrus kandleri. In-

crease of catalytic eciency (kt/km) and thermostability in

the presence of salts. Arch. Microbiol. 156, 517^524.

[12] Bult, C.J., White, O., Olsen, G.J., Zhou, L.X., Fleischmann,

R.D., Sutton, G.G., Blake, J.A., Fitzgerald, L.M., Clayton,

R.A., Gocayne, J.D., Kerlavage, A.R., Dougherty, B.A.,

Tomb, J.F., Adams, M.D., Reich, C.I., Overbeek, R., Kirk-

ness, E.F., Weinstock, K.G., Merrick, J.M., Glodek, A.,

Scott, J.L., Geoghagen, N.S.M., Weidman, J.F., Fuhrmann,

J.L., Nguyen, D., Utterback, T.R., Kelley, J.M., Peterson,J.D., Sadow, P.W., Hanna, M.C., Cotton, M.D., Roberts,

K.M., Hurst, M.A., Kaine, B.P., Borodovsky, M., Klenk,

H.P., Fraser, C.M., Smith, H.O., Woese, C.R. and Venter,

J.C. (1996) Complete genome sequence of the methanogenic

archaeon, Methanococcus jannaschii. Science 273, 1058^

1073.

[13] Burke, S.A. and Krzycki, J.A. (1995) Involvement of the `A'

isozyme of methyltransferase II and the 29-kilodalton corri-

noid protein in methanogenesis from monomethylamine.

J. Bacteriol. 177, 4410^4416.

[14] Burke, S.A. and Krzycki, J.A. (1997) Reconstitution of mono-

methylamine:coenzyme M methyl transfer with a corrinoid

protein and two methyltransferases puried from Methanosar-

cina barkeri. J. Biol. Chem. 272, 16570^16577.[15] Burke, S.A., Lo, S.L. and Krzycki, J.A. (1998) Clustered

genes encoding the methyltransferases of methanogenesis

from monomethylamine. J. Bacteriol. 180, 3432^3440.

[16] Buss, K.A., Ingram-Smith, C., Ferry, J.G., Sanders, D.A. and

Hasson, M.S. (1997) Crystallization of acetate kinase from

Methanosarcina thermophila and prediction of its fold. Protein

Sci. 6, 2659^2662.

[17] Chistoserdova, L., Vorholt, J.A., Thauer, R.K. and Lidstrom,

M.E. (1998) C-1 transfer enzymes and coenzymes linking

methylotrophic bacteria and methanogenic Archaea. Science

281, 99^102.

[18] Cram, D., Sherf, S.B., Libby, A.R., Mattaliano, T.R., Ram-

achandran, J.K. and Reeve, J.N. (1987) Structure and expres-

sion of the genes, mcrBDCGA, which encode the subunits of

component C of methyl coenzyme M reductase in Methano-

coccus vannielii. Proc. Natl. Acad. Sci. USA 84, 3992^

3996.

[19] Daas, P.J.H., Gerrits, K.A.A., Keltjens, J.T., Vanderdrift, C.

and Vogels, G.D. (1993) Involvement of an activation protein

in the methanol:2-mercaptoethanesulfonic acid methyltrans-

ferase reaction in Methanosarcina barkeri. J. Bacteriol. 175,

1278^1283.

[20] Daas, P.J.H., Hagen, W.R., Keltjens, J.T., Vanderdrift, C.

and Vogels, G.D. (1996) Activation mechanism of metha-

nol:5- hydroxybenzimidazolylcobamide methyltransferase

from Methanosarcina barkeri. J. Biol. Chem. 271, 22346^

22351.

[21] Daas, P.J.H., Wassenaar, R.W., Willemsen, P., Theunissen,

R.J., Keltjens, J.T., Vanderdrift, C. and Vogels, G.D. (1996)

Purication and properties of an enzyme involved in the ATP-

dependent activation of the methanol:2-mercaptoethanesul-

fonic acid methyltransferase reaction in Methanosarcina bar-

keri. J. Biol. Chem. 271, 22339^22345.

[22] Deppenmeier, U., Blaut, M., Jussoe, A. and Gottschalk, G.

(1988) A methyl-CoM methylreductase system from methano-

genic bacterium strain Go1 not requiring ATP for activity.

FEBS Lett. 241, 60^64.

[23] Deppenmeier, U., Muller, V. and Gottschalk, G. (1996) Path-

ways of energy conservation in methanogenic archaea. Arch.

Microbiol. 165, 149^163.

[24] DiMarco, A., Donnelly, A.M. and Wolfe, R.S. (1986) Puri-

cation and properties of the 5,10-methenyltetrahydrometha-

nopterin cyclohydrolase from Methanobacterium thermoauto-trophicum. J. Bacteriol. 168, 1372^1377.

[25] DiMarco, A., Sment, A.K., Konisky, A.J. and Wolfe, R.S.

(1990) The formylmethanofuran:tetrahydromethanopterin

formyltransferase from Methanobacterium thermoautotrophi-

cum. Nucleotide sequence and functional expression of the

cloned gene. J. Biol. Chem. 265, 472^476.

[26] Drennan, C., Huang, S., Drummond, J.T., Matthews, R.G.

and Ludwig, M.L. (1994) How a protein binds B12, a 3.0 A

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

thase. Science 266, 1669^1674.

[27] Eggen, R.I.L., Vankranenburg, R., Vriesema, A.J.M., Geerl-

ing, A.C.M., Verhagen, M.F.J.M., Hagen, W.R. and Devos,

W.M. (1996) Carbon monoxide dehydrogenase from Metha-

nosarcina frisia Go1. Characterization of the enzyme and theregulated expression of two operon-like cdh gene clusters.

J. Biol. Chem. 271, 14256^14263.

[28] Ellermann, J., Rospert, S., Thauer, R.K., Bokranz, M., Klein,

A., Voges, M. and Berkessel, A. (1989) Methyl-coenzyme M

reductase from Methanobacterium thermoautotrophicum

(strain Marburg): purity, activity and novel inhibitors. Eur.

J. Biochem. 184, 63^68.

[29] Enssle, M., Zirngibl, C., Linder, D. and Thauer, R.K. (1991)

Coenzyme FRPH dependent NS, NIH- methylenetetrahydrome-

thanopterin dehydrogenase in methanol grown Methanosarci-

na barkeri. Arch. Microbiol. 155, 483^490.

[30] Ermler, U., Grabarse, W., Shima, S., Goubeaud, M. and

Thauer, R.K. (1997) Crystal structure of methyl-coenzyme

M reductase: the key enzyme of biological methane forma-

tion. Science 278, 1457^1462.

[31] Ermler, U., Merckel, M., Thauer, R. and Shima, S. (1997)

Formylmethanofuran: tetrahydromethanopterin formyltrans-

ferase from Methanopyrus kandleri ^ new insights into salt-

dependence and thermostability. Structure 5, 635^646.

[32] Ferguson, D.J. and Krzycki, J.A. (1997) Reconstitution of


8/6/2019 Fems Rev v23 p13

22/26

trimethylamine-dependent coenzyme M methylation with the

trimethylamine corrinoid protein and the isozymes of methyl-

transferase II from Methanosarcina barkeri. J. Bacteriol. 179,

846^852.

[33] Ferguson, D.J., Jr., Krzycki, J.A. and Grahame, D.A. (1996)

Specic roles of methylcobamide:coenzyme M methyltransfer-

ase isozymes in metabolism of methanol and methylamines in

Methanosarcina barkeri. J. Biol. Chem. 271, 5189^5194.

[34] Ferry, J.G. (1993) Biochemistry of Methanogenesis. Chapman

and Hall, New York.

[35] Ferry, J.G. (1997) Enzymology of the fermentation of acetate

to methane by Methanosarcina thermophila. BioFactors 6, 25^

35.

[36] Gartner, P., Ecker, A., Fischer, R., Linder, D., Fuchs, G. and

Thauer, R.K. (1993) Purication and properties of NS- meth-

yltetrahydromethanopterin:coenzyme M methyltransferase

from Methanobacterium thermoautotrophicum. Eur. J. Bio-

chem. 213, 537^545.

[37] Gartner, P., Weiss, D.S., Harms, U. and Thauer, R.K. (1994)

NS-Methyltetrahydromethanopterin:coenzyme M methyl-

transferase from Methanobacterium thermoautotrophicum Cat-

alytic mechanism and sodium ion dependence. Eur. J. Bio-

chem. 226, 465^472.[38] Goubeaud, M., Schreiner, G. and Thauer, R.K. (1997) Puri-

ed methyl-coenzyme-M reductase is activated when the en-

zyme-bound coenzyme FRQH is reduced to the nickel(I) oxida-

tion state by titanium(III) citrate. Eur. J. Biochem. 243, 110^

114.

[39] Grahame, D.A. (1993) Substrate and cofactor reactivity of a

carbon monoxide dehydrogenase corrinoid enzyme complex.

Stepwise reduction of iron sulfur and corrinoid centers, the

corrinoid CoPaI redox midpoint potential, and overall syn-

thesis of acetyl-CoA. Biochemistry 32, 10786^10793.

[40] Grahame, D.A. and Demoll, E. (1996) Partial reactions cata-

lyzed by protein components of the acetyl-CoA decarbonylase

synthase enzyme complex from Methanosarcina barkeri.

J. Biol. Chem. 271, 8352^8358.[41] Grahame, D.A. and Demoll, E. (1995) Substrate and acces-

sory protein requirements and thermodynamics of acetyl-CoA

synthesis and cleavage in Methanosarcina barkeri. Biochemis-

try 34, 4617^4624.

[42] Grahame, D.A., Khangulov, S. and Demoll, E. (1996) Reac-

tivity of a paramagnetic enzyme-CO adduct in acetyl-CoA

synthesis and cleavage. Biochemistry 35, 593^600.

[43] Harms, U. and Tauer, R.K. (1996) The corrinoid-containing

23-kDa subunit MtrA of the energy- conserving NS-methylte-

trahydromethanopterin: coenzyme M methyltransferase com-

plex from Methanobacterium thermoautotrophicum EPR spec-

troscopic evidence for a histidine residue as a cobalt ligand of

the cobamide. Eur. J. Biochem. 241, 149^154.

[44] Harms, U. and Thauer, R.K. (1996) Methylcobalamin:coen-

zyme M methyltransferase isoenzymes MtaA and MtbA from

Methanosarcina barkeri. Cloning, sequencing and dierential

transcription of the encoding genes, and functional overex-

pression of the MtaA gene in Escherichia coli. Eur. J. Bio-

chem. 235, 653^659.

[45] Harms, U., Weiss, D.S., Gartner, P., Linder, D. and Thauer,

R.K. (1995) The energy conserving NS-methyltetrahydrometh-

anopterin: coenzyme M methyltransferase complex from

Methanobacterium thermoautotrophicum is composed of eight

dierent subunits. Eur. J. Biochem. 228, 640^648.

[46] Hartmann, G.C., Klein, A.R., Linder, M. and Thauer, R.K.

(1996) Purication, properties and primary structure of HP-

forming NS,NIH-methylenetetrahydromethanopterin dehydro-

genase from Methanococcus thermolithotrophicus. Arch. Mi-

crobiol. 165, 187^193.

[47] Hausinger, R.P., Orme-Johnson, W.H. and Walsh, C. (1985)

Factor 390 chromophores: phosphodiester between AMP or

GMP and methanogen Factor 420. Biochemistry 24, 1629^

1633.

[48] Henkin, J. and Abeles, R.H. (1976) Evidence against an acyl-

enzyme intermediate in the reaction catalyzed by clostridial

phosphotransacetylase. Biochemistry 15, 3472^3479.

[49] Hochheimer, A., Linder, D., Thauer, R.K. and Hedderich, R.

(1996) The molybdenum formylmethanofuran dehydrogenase

operon and the tungsten formylmethanofuran dehydrogenase

operon from Methanobacterium thermoautotrophicum ^ struc-

tures and transcriptional regulation. Eur. J. Biochem. 242,

156^162.

[50] Hochheimer, A., Schmitz, R.A., Thauer, R.K. and Hedderich,R. (1995) The tungsten formylmethanofuran dehydrogenase

from Methanobacterium thermoautotrophicum contains se-

quence motifs characteristic for enzymes containing molyb-

dopterin dinucleotide. Eur. J. Biochem. 234, 910^920.

[51] Hu, Z.G., Spangler, N.J., Anderson, M.E., Xia, J.Q., Ludden,

P.W., Lindahl, P.A. and M.E. (1996) Nature of the C-cluster

in Ni-containing carbon monoxide dehydrogenases. J. Am.

Chem. Soc. 118, 830^845.

[52] Huber, C. and Wachtershauser, G. (1997) Activated acetic

acid by carbon xation on (Fe,Ni)S under primordial condi-

tions. Science 276, 245^247.

[53] Jablonski, P.E. and Ferry, J.G. (1991) Purication and prop-

erties of methyl coenzyme M methylreductase from acetate

grown Methanosarcina thermophila. J. Bacteriol. 173, 2481^2487.

[54] Jablonski, P.E., Lu, W.P., Ragsdale, S.W. and Ferry, J.G.

(1993) Characterization of the metal centers of the corri-

noid/iron- sulfur component of the CO dehydrogenase enzyme

complex from Methanosarcina thermophila by EPR spectro-

scopy and spectroelectrochemistry. J. Biol. Chem. 268, 325^

329.

[55] Jaun, B. (1993) Methane Formation by Methanogenic Bacte-

ria: Redox Chemistry of Coenzyme FRQH, Vol. 29. Marcel

Dekker, New York.

[56] Jetten, M.S.M., Hagen, W.R., Pierik, A.J., Stams, A.J.M. and

Zehnder, A.J.B. (1991) Paramagnetic centers and acetyl-coen-

zyme A/CO exchange activity of carbon monoxide dehydro-

genase from Methanothrix soehngenii. Eur. J. Biochem. 195,

385^391.

[57] Jetten, M.S.M., Stams, A.J.M. and Zehnder, A.J.B. (1990)

Purication and some properties of the methyl-CoM reductase

of Methanothrix soehngenii. FEMS Microbiol. Lett. 66, 183^

186.

[58] Johnson, J.L., Bastian, N.R., Schauer, N.L., Ferry, J.G. and


8/6/2019 Fems Rev v23 p13

23/26

Rajagopalan, K.V. (1991) Identication of molybdopterin

guanine dinucleotide in formate dehydrogenase from Metha-

nobacterium formicicum. FEMS Microbiol. Lett. 77, 2^3.

[59] Karrasch, M., Borner, G., Enssle, M. and Thauer, R.K.

(1989) Formylmethanofuran dehydrogenase from methano-

genic bacteria, a molybdoenzyme. FEBS Lett. 253, 226^230.

[60] Karrasch, M., Borner, G., Enssle, M. and Thauer, R.K.

(1990) The molybdoenzyme formylmethanofuran dehydrogen-

ase from Methanosarcina barkeri contains a pterin cofactor.

Eur. J. Biochem. 194, 367^372.

[61] Karrasch, M., Borner, G. and Thauer, R.K. (1990) The mo-

lybdenum cofactor of formylmethanofuran dehydrogenase

from Methanosarcina barkeri is a molybdopterin guanine di-

nucleotide. FEBS Lett. 274, 48^52.

[62] Kengen, S.W.M., Daas, P.J.H., Duits, E.F.G., Keltjens, J.T.,

Vanderdrift, C. and Vogels, G.D. (1992) Isolation of a 5-hy-

droxybenzimidazolyl cobamide-containing enzyme involved in

the methyltetrahydromethanopterin-coenzyme M methyltrans-

ferase reaction in Methanobacterium thermoautotrophicum. Bi-

ochim. Biophys. Acta 1118, 249^260.

[63] Kerby, R.L., Hong, S.S., Ensign, S.A., Coppoc, L.J., Ludden,

P.W. and Roberts, G.P. (1992) Genetic and physiological

characterization of the Rhodospirillum rubrum carbon monox-ide dehydrogenase system. J. Bacteriol. 174, 5284^5294.

[64] Kerby, R.L., Ludden, P.W. and Roberts, G.P. (1997) In vivo

nickel insertion into the carbon monoxide dehydrogenase of

Rhodospirillum rubrum : Molecular and physiological charac-

terization of cooCTJ. J. Bacteriol. 179, 2259^2266.

[65] Kisker, C., Schindelin, H., Alber, B.E., Ferry, J.G. and Rees,

D.C. (1996) A left-handed beta-helix revealed by the crystal

structure of a carbonic anhydrase from the archaeon Metha-

nosarcina thermophila. EMBO J. 15, 2323^2330.

[66] Klein, A., Allmansberger, R., Bokranz, M., Knaub, S., Mul-

ler, B. and Muth, E. (1988) Comparative analysis of genes

encoding methyl coenzyme M reductase in methanogenic bac-

teria. Mol. Gen. Genet. 213, 409^420.

[67] Klein, A.R., Fernandez, V.M. and Thauer, R.K. (1995) HP-forming NS,NIH-methylenetetrahydromethanopterin dehydro-

genase: mechanism of HP formation analyzed using hydrogen

isotopes. FEBS Lett. 368, 203^206.

[68] Klein, A.R., Hartmann, G.C. and Thauer, R.K. (1995) Hy-

drogen isotope eects in the reactions catalyzed by HP-form-

ing NS,NIH-methylenetetrahydromethanopterin dehydrogenase

from methanogenic archaea. Eur. J. Biochem. 233, 372^

376.

[69] Klein, A.R., Koch, J., Stetter, K.O. and Thauer, R.K. (1993)

Two NS,NIH-methylenetetrahydromethanopterin dehydrogen-

ases in the extreme thermophile Methanopyrus kandleri: char-

acterization of the coenzyme F420-dependent enzyme. Arch.

Microbiol. 160, 186^192.

[70] Klein, A.R. and Thauer, R.K. (1997) Overexpression of the

coenzyme FRPH-dependent NS,NIH- methylenetetrahydrometha-

nopterin dehydrogenase gene from the hyperthermophilic

Methanopyrus kandleri. Eur. J. Biochem. 245, 386^391.

[71] Klein, A.R. and Thauer, R.K. (1995) Re-face specicity at

C14a of methylenetetrahydromethanopterin and Si-face spe-

cicity at C5 of coenzyme FRPH for coenzyme FRPH-dependent

methylenetetrahydromethanopterin dehydrogenase from

methanogenic archaea. Eur. J. Biochem. 227, 169^174.

[72] Koonin, E.V., Mushegian, A.R., Galperin, M.Y. and Walker,

D.R. (1997) Comparison of archaeal and bacterial genomes:

computer analysis of protein sequences predicts novel func-

tions and suggests a chimeric origin for the archaea. Mol.

Microbiol. 25, 619^637.

[73] Kunow, J., Shima, S., Vorholt, J.A. and Thauer, R.K. (1996)

Primary structure and properties of the formyltransferase

from the mesophilic Methanosarcina barkeri: comparison

with the enzymes from thermophilic and hyperthermophilic

methanogens. Arch. Microbiol. 165, 97^105.

[74] Latimer, M.T. and Ferry, J.G. (1993) Cloning, sequence anal-

ysis, and hyperexpression of the genes encoding phosphotrans-

acetylase and acetate kinase from Methanosarcina thermophila.

J. Bacteriol. 175, 6822^6829.

[75] Leclerc, G.M. and Grahame, D.A. (1996) Methylcobamide:

coenzyme M methyltransferase isozymes from Methanosarcina

barkeri. Physicochemical characterization, cloning, sequence

analysis and heterologous gene expression. J. Biol. Chem.

271, 18725^18731.

[76] Lehmacher, A. (1994) Cloning, sequencing and transcript

analysis of the gene encoding formylmethanofuran:tetrahy-dromethanopterin formyltransferase from the hyperthermo-

philic Methanothermus fervidus. Mol. Gen. Genet. 242, 73^

80.

[77] Lehmacher, A. and Klenk, H.P. (1994) Characterization and

phylogeny of mcrII, a gene cluster encoding an isoenzyme of

methyl coenzyme M reductase from hyperthermophilic Meth-

anothermus fervidus. Mol. Gen. Genet. 243, 198^206.

[78] Lienard, T., Becher, B., Marschall, M., Bowien, S. and Gott-

schalk, G. (1996) Sodium ion translocation by NS-methylte-

trahydromethanopterin: coenzyme M methyltransferase from

Methanosarcina mazei Go1 reconstituted in ether lipid lipo-

somes. Eur. J. Biochem. 239, 857^864.

[79] Lienard, T. and Gottschalk, G. (1998) Cloning, sequencing

and expression of the genes encoding the sodium translocatingNS-methyltetrahydromethanopterin:coenzyme M methyl-

transferase of the methylotrophic archaeon Methanosarcina

mazei Go1. FEBS Lett. 425, 204^208.

[80] Lu, W.P., Becher, B., Gottschalk, G. and Ragsdale, S.W.

(1995) Electron paramagnetic resonance spectroscopic and

electrochemical characterization of the partially puried N S-

methyltetrahydromethanopterin:coenzyme M methyltransfer-

ase from Methanosarcina mazei Go1. J. Bacteriol. 177,

2245^2250.

[81] Lu, W.P., Jablonski, P.E., Rasche, M., Ferry, J.G. and Rags-

dale, S.W. (1994) Characterization of the metal centers of the

Ni-Fe-S component of the carbon-monoxide dehydrogenase

enzyme complex from Methanosarcina thermophila. J. Biol.

Chem. 269, 9736^9742.

[82] Ludwig, M.L. and Matthews, R.G. (1997) Structure-based

perspectives on BIP-dependent enzymes. Annu. Rev. Biochem.

66, 269^313.

[83] Ma, K., Linder, D., Stetter, K.O. and Thauer, R.K. (1991)

Purication and properties of NS,NIH- methylenetetrahydro-

methanopterin reductase (coenzyme FRPH-dependent) from


8/6/2019 Fems Rev v23 p13

24/26

the extreme thermophile Methanopyrus kandleri. Arch. Micro-

biol. 155, 593^600.

[84] Ma, K. and Thauer, R.K. (1990) Purication and properties

of NS,NIH- methylenetetrahydromethanopterin reductase from

Methanobacterium thermoautotrophicum (strain Marburg).

Eur. J. Biochem. 191, 187^193.

[85] Ma, K. and Thauer, R.K. (1990) Single step purication of

methylenetetrahydromethanopterin reductase from Methano-

bacterium thermoautotrophicum by specic binding to Blue Se-

pharose Cl-6B. FEBS Lett. 268, 59^62.

[86] Ma, K., Zirngibl, C., Linder, D., Stetter, K.O. and Thauer,

R.K. (1991) NS, NIH-methylenetetrahydromethanopterin dehy-

drogenase (HP- forming) from the extreme thermophile Meth-

anopyrus kandleri. Arch. Microbiol. 156, 43^48.

[87] Ma, K.S. and Thauer, R.K. (1990) NS, NIH-methylenetetrahy-

dromethanopterin reductase from Methanosarcina barkeri.

FEMS Microbiol. Lett. 70, 119^124.

[88] Maupin-Furlow, J.A., and Ferry, J.G. (1996) Analysis of the

CO dehydrogenase/acetyl-coenzyme A synthase operon of

Methanosarcina thermophila. J. Bacteriol. 178, 6849^6856.

[89] Morgan, R.M., Pihl, T.D., Nolling, J. and Reeve, J.N. (1997)

Hydrogen regulation of growth, growth yields, and methane

gene transcription in Methanobacterium thermoautotrophicumdelta H. J. Bacteriol. 179, 889^898.

[90] Morton, T.A., Runquist, J.A., Ragsdale, S.W., Shanmugasun-

daram, T., Wood, H.G. and Ljungdahl, L.G. (1991) The Pri-

mary Structure of the subunits of carbon monoxide dehydro-

genase/acetyl-CoA synthase from Clostridium thermoaceticum.

J. Biol. Chem. 266, 23824^23828.

[91] Mukhopadhyay, B. and Daniels, L. (1989) Aerobic purica-

tion of NS, NIH methylenetetrahydromethanopterin dehydro-

genase, separated from NS, NIH methenyltetrahydromethanop-

terin cyclohydrolase, from Methanobacterium thermoauto-

trophicum strain Marburg. Can. J. Microbiol. 35, 499^507.

[92] Mukhopadhyay, B., Purwantini, E., Pihl, T.D., Reeve, J.N.

and Daniels, L. (1995) Cloning, sequencing, and transcription-

al analysis of the coenzyme FRPH-dependent methylene-5,6,7,8-tetrahydromethanopterin dehydrogenase gene from Methano-

bacterium thermoautotrophicum strain Marburg and functional

expression in Escherichia coli. J. Biol. Chem. 270, 2827^

2832.

[93] Nolling, J., Pihl, T.D. and Reeve, J.N. (1995) Cloning, se-

quencing, and growth phase-dependent transcription of the

coenzyme FRPH-dependent NS,NIH- methylenetetrahydrometha-

fems rev v23 p13

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