Methylthiol:coenzyme M methyltransferase from Methanosarcina barkeri, an enzyme of methanogenesis from dimethylsulfide and methylmercaptopropionate.

During growth on acetate, Methanosarcina barkeri expresses catabolic enzymes for other methanogenic substrates such as monomethylamine. The range of substrates used by cells grown on acetate was further explored, and it was found that cells grown on acetate also converted dimethylsulfide (DMS) and methylmercaptopropionate (MMPA) to methane. Cells or extracts of cells grown on trimethylamine or methanol did not utilize either DMS or MMPA. During growth on acetate, cultures demethylated MMPA, producing methane and mercaptopropionate. Extracts of acetate-grown cells possessed DMS- and MMPA-dependent coenzyme M (CoM) methylation activities. The activity peaks of CoM methylation with either DMS or MMPA coeluted upon gel permeation chromatography of extracts of acetate-grown cells consistent with an apparent molecular mass of 470 kDa. A 480-kDa corrinoid protein, previously demonstrated to be a CoM methylase but otherwise of unknown physiological function, was found to methylate CoM with either DMS or MMPA. MMPA was demethylated by the purified 480-kDa CoM methylase, consuming 1 mol of CoM and producing 1 mol of mercaptopropionate. DMS was demethylated by the purified protein, consuming 1 mol of CoM and producing 1 mol of methanethiol. The methylthiol:CoM methyltransferase reaction could be initiated only with the enzyme-bound corrinoid in the methylated state. CoM could demethylate, and DMS and MMPA could remethylate, the corrinoid cofactor. The monomethylamine corrinoid protein and the A isozyme of methylcobamide:CoM methyltransferase (proteins homologous to the two subunits comprising the 480-kDa CoM methylase) did not catalyze CoM methylation with methylated thiols. These results indicate that the 480-kDa corrinoid protein functions as a CoM methylase during methanogenesis from DMS or MMPA.     

Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.

Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction.     

Isolation of two novel corrinoid proteins from acetate-grown Methanosarcina barkeri.

Two corrinoid proteins with molecular sizes of 480 and 29 kDa are stably methylated by [2-14C]acetate-derived intermediates in cell extracts of aceticlastic Methanosarcina barkeri when methylreductase is inhibited by the addition of bromoethanesulfonic acid. Both 14CH3-proteins have been isolated to near homogeneity and found to be abundant soluble proteins. The larger protein possesses two subunits, of 41.4 and 30.4 kDa, in an equimolar ratio, suggesting an alpha 6 beta 6 conformation with six bound methylated corrinoids per 480-kDa molecule. The 29-kDa protein is a monomer in solution and possesses only one methylated corrinoid. All methyl groups on both proteins are photolabile, but the methylated corrinoid bound to the 29-kDa protein undergoes photolysis at a higher rate than that bound to the 480-kDa protein. The two proteins possess discrete N termini and do not appear to be forms of the same protein in equilibrium. Neither protein has an Fe4S4 cluster, and both have UV-visible spectra most similar to that of a base-on methylated corrinoid. A previously identified methylated protein, designated the unknown A 14CH3-protein, copurifies with the 480-kDa protein and has the same subunit composition. The methyl groups of both isolated 14CH3-proteins are converted to methane in cell extracts. The methylated proteins that accumulate in extracts in the presence of bromoethanesulfonic acid are demethylated by the addition of coenzyme M. Both isolated proteins are abundant novel corrinoid proteins that can methylate and be methylated by intermediates of the methanogenic pathway.     

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO(2)/H(2), CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl-CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol-induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537-540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co-transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X-ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the alpha-axial ligand replacing benziimidazole, suggesting base-off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica.     

Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.

Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.     

The MtsA subunit of the methylthiol:coenzyme M methyltransferase of Methanosarcina barkeri catalyses both half-reactions of corrinoid-dependent dimethylsulfide: coenzyme M methyl transfer

Methanogenesis from dimethylsulfide requires the intermediate methylation of coenzyme M. This reaction is catalyzed by a methylthiol:coenzyme M methyltransferase composed of two polypeptides, MtsA (a methylcobalamin:coenzyme M methyltransferase) and MtsB (homologous to a class of corrinoid proteins involved in methanogenesis). Recombinant MtsA was purified and found to be a homodimer that bound one zinc atom per polypeptide, but no corrinoid cofactor. MtsA is an active methylcobalamin:coenzyme M methyltransferase, but also methylates cob(I)alamin with dimethylsulfide, yielding equimolar methylcobalamin and methanethiol in an endergonic reaction with a K(eq) of 5 x 10(-)(4). MtsA and cob(I)alamin mediate dimethylsulfide:coenzyme M methyl transfer in the complete absence of MtsB. Dimethylsulfide inhibited methylcobalamin:coenzyme methyl transfer by MtsA. Inhibition by dimethylsulfide was mixed with respect to methylcobalamin, but competitive with coenzyme M. MtbA, a MtsA homolog participating in coenzyme M methylation with methylamines, was not inhibited by dimethylsulfide and did not catalyze detectable dimethylsulfide:cob(I)alamin methyl transfer. These results are most consistent with a model for the native methylthiol:coenzyme M methyltransferase in which MtsA mediates the methylation of corrinoid bound to MtsB with dimethylsulfide and subsequently demethylates MtsB-bound corrinoid with coenzyme M, possibly employing elements of the same methyltransferase active site for both reactions.     

Reconstitution of dimethylamine:coenzyme M methyl transfer with a discrete corrinoid protein and two methyltransferases purified from Methanosarcina barkeri

Methyl transfer from dimethylamine to coenzyme M was reconstituted in vitro for the first time using only highly purified proteins. These proteins isolated from Methanosarcina barkeri included the previously unidentified corrinoid protein MtbC, which copurified with MtbA, the methylcorrinoid:Coenzyme M methyltransferase specific for methanogenesis from methylamines. MtbC binds 1.0 mol of corrinoid cofactor/mol of 24-kDa polypeptide and stimulated dimethylamine:coenzyme M methyl transfer 3.4-fold in a cell extract. Purified MtbC and MtbA were used to assay and purify a dimethylamine:corrinoid methyltransferase, MtbB1. MtbB1 is a 230-kDa protein composed of 51-kDa subunits that do not possess a corrinoid prosthetic group. Purified MtbB1, MtbC, and MtbA were the sole protein requirements for in vitro dimethylamine:coenzyme M methyl transfer. An MtbB1:MtbC ratio of 1 was optimal for coenzyme M methylation with dimethylamine. MtbB1 methylated either corrinoid bound to MtbC or free cob(I)alamin with dimethylamine, indicating MtbB1 carries an active site for dimethylamine demethylation and corrinoid methylation. Experiments in which different proteins of the resolved monomethylamine:coenzyme M methyl transfer reaction replaced proteins involved in dimethylamine:coenzyme M methyl transfer indicated high specificity of MtbB1 and MtbC in dimethylamine:coenzyme M methyl transfer activity. These results indicate MtbB1 demethylates dimethylamine and specifically methylates the corrinoid prosthetic group of MtbC, which is subsequently demethylated by MtbA to methylate coenzyme M during methanogenesis from dimethylamine.     

Acetate-dependent methylation of two corrinoid proteins in extracts of Methanosarcina barkeri.

Corrinoid proteins have been implicated as methyl carriers in methane formation from acetate, yet specific corrinoid proteins methylated by acetate-derived intermediates have not been identified. In the presence of ATP, H2, and bromoethanesulfonic acid, label from 3H- or 2-14C-labeled acetate was incorporated into the protein fraction of cell extracts of Methanosarcina barkeri. Incorporated label was susceptible to photolysis, yielding labeled methane as the anaerobic photolysis product. Size exclusion high-pressure liquid chromatography (HPLC) demonstrated the presence of at least three labeled proteins with native molecular sizes of 480, 200, and 29 kDa, while electrophoresis indicated that four major labeled proteins were present. Dual-label experiments demonstrated that these four proteins were methylated rather than acetylated. Two of the proteins (480 and 29 kDa) contained the majority of radiolabel and were stably methylated. After labeling with [2-14C]acetate, the stable 14CH3-proteins were partially purified, and 14CH3-cofactors were isolated from each protein. UV-visible spectroscopy and HPLC demonstrated these to be methylated corrinoids. When the 480-kDa corrinoid protein was purified to 70% homogeneity, the preparation was found to have subunits of 40 and 30 kDa. The 480-kDa protein but not the 29-kDa protein was methylated during in vitro methanogenesis from acetate and demethylated as methanogenesis ceased, consistent with the involvement of this protein in methane formation.     

Tetramethylammonium:coenzyme M methyltransferase system from Methanococcoides sp.

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens. Received: 7 April 1998 / Accepted: 26 June 1998     

Differential in vitro methylation and synthesis of the 480-kilodalton corrinoid protein in Methanosarcina barkeri grown on different substrates.

The 480-kDa corrinoid protein was significantly methylated in extracts of acetate- but not methanol-grown cells incubated with 14CH3OH, in part because of its decreased synthesis in cells grown on substrates other than acetate. In addition, a 200-kDa corrinoid protein was methylated in extracts of methanol- but not acetate-grown cells.     

Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.     

Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro.

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.     

Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.

The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of chloromethane to tetrahydrofolate, thus affording a direct link between chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.     

The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH.

In methanogenic archaea the transfer of the methyl group of N5-methyltetrahydromethanopterin to coenzyme M is coupled with energy conservation. The reaction is catalyzed by a membrane associated multienzyme complex composed of eight different subunits MtrA-H. The 23 kDa subunit MtrA harbors a corrinoid prosthetic group which is methylated and demethylated in the catalytic cycle. We report here that the 34 kDa subunit MtrH catalyzes the methylation reaction. MtrH was purified and shown to exhibit methyltetrahydromethanopterin:cob(I)alamin methyltransferase activity. Sequence comparison revealed similarity of MtrH with MetH from Escherichia coli and AcsE from Clostridium thermoaceticum: both enzymes exhibit methyltetrahydrofolate:cob(I)alamin methyltransferase activity.     

RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.Most methanogenic Archaea are capable of producing methane only from carbon dioxide. The Methanosarcinaceae are a notable exception as representatives are capable of methylotrophic methanogenesis from methylated amines, methylated thiols, or methanol. Methanogenesis from these substrates requires methylation of 2-mercaptoethanesulfonic acid (coenzyme M or CoM) that is subsequently used by methylreductase to generate methane and a mixed disulfide whose reduction leads to energy conservation (1–4).Methylation of CoM with trimethylamine (TMA),⁴ dimethylamine (DMA), or monomethylamine (MMA) is initiated by three distinct methyltransferases that methylate cognate corrinoid-binding proteins (3). MtmB, the MMA methyltransferase, specifically methylates cognate corrinoid protein, MtmC, with MMA (see Fig. 1) (5, 6). The DMA methyltransferase, MtbB, and its cognate corrinoid protein, MtbC, interact specifically to demethylate DMA (7, 8). TMA is demethylated by the TMA methyltransferase (MttB) in conjunction with the TMA corrinoid protein (MttC) (8, 9). Each of the methylated corrinoid proteins is a substrate for a methylcobamide:CoM methyltransferase, MtbA, which produces methyl-CoM (10–12).Open in a separate windowFIGURE 1.MMA:CoM methyl transfer. A schematic of the reactions catalyzed by MtmB, MtmC, and MtbA is shown that emphasizes the key role of MtmC in the catalytic cycle of both methyltransferases. Oxidation to Co(II)-MtmC of the supernucleophilic Co(I)-MtmC catalytic intermediate inactivates methyl transfer from MMA to the thiolate of coenzyme M (HSCoM). In vitro reduction of the Co(II)-MtmC with either methyl viologen reduced to the neutral species or with RamA in an ATP-dependent reaction can regenerate the Co(I) species. In either case in vitro Ti(III)-citrate is the ultimate source of reducing power.CoM methylation with methanol requires the methyltransferase MtaB and the corrinoid protein MtaC, which is then demethylated by another methylcobamide:CoM methyltransferase, MtaA (13–15). The methylation of CoM with methylated thiols such as dimethyl sulfide in Methanosarcina barkeri is catalyzed by a corrinoid protein that is methylated by dimethyl sulfide and demethylated by CoM, but in this case an associated CoM methylase carries out both methylation reactions (16).In bacteria, analogous methyltransferase systems relying on small corrinoid proteins are used to achieve methylation of tetrahydrofolate. In Methylobacterium spp., CmuA, a single methyltransferase with a corrinoid binding domain, along with a separate pterin methylase, effect the methylation of tetrahydrofolate with chloromethane (17, 18). In Acetobacterium dehalogenans and Moorella thermoacetica various three-component systems exist for specific demethylation of different phenylmethyl ethers, such as vanillate (19) and veratrol (20), again for the methylation of tetrahydrofolate. Sequencing of the genes encoding the corrinoid proteins central to the archaeal and bacterial methylotrophic pathways revealed they are close homologs. Furthermore, genes predicted to encode such corrinoid proteins and pterin methyltransferases are widespread in bacterial genomes, often without demonstrated metabolic function. All of these corrinoid proteins are similar to the well characterized cobalamin binding domain of methionine synthase (21, 22).In contrast, the TMA, DMA, MMA, and methanol methyltransferases are not homologous proteins. The methylamine methyltransferases do share the common distinction of having in-frame amber codons (6, 8) within their encoding genes that corresponds to the genetically encoded amino acid pyrrolysine (23–25). Pyrrolysine has been proposed to act in presenting a methylammonium adduct to the central cobalt ion of the corrinoid protein for methyl transfer (3, 23, 26). However, nucleophilic attack on a methyl donor requires the central cobalt ion of a corrinoid cofactor is in the nucleophilic Co(I) state rather than the inactive Co(II) state (27). Subsequent demethylation of the methyl-Co(III) corrinoid cofactor regenerates the nucleophilic Co(I) cofactor. The Co(I)/Co(II) in the cobalamin binding domain of methionine synthase has an E_m value of -525 mV at pH 7.5 (28). It is likely to be similarly low in the homologous methyltrophic corrinoid proteins. These low redox potentials make the corrinoid cofactor subject to adventitious oxidation to the inactive Co(II) state (Fig. 1).During isolation, these corrinoid proteins are usually recovered in a mixture of Co(II) or hydroxy-Co(III) states. For in vitro studies, chemical reduction can maintain the corrinoid protein in the active Co(I) form. The methanol:CoM or the phenylmethyl ether:tetrahydrofolate methyltransferase systems can be activated in vitro by the addition of Ti(III) alone as an artificial reductant (14, 19). In contrast, activation of the methylamine corrinoid proteins further requires the addition of methyl viologen as a redox mediator. Ti(III) reduces methyl viologen to the extremely low potential neutral species. In vitro activation with these agents does not require ATP (5, 7, 9).Cellular mechanisms also exist to achieve the reductive activation of corrinoid cofactors in methyltransferase systems. Activation of human methionine synthase involves reduction of the co(II)balamin by methionine synthase reductase (29), whereas the Escherichia coli enzyme requires flavodoxin (30). The endergonic reduction is coupled with the exergonic methylation of the corrinoid with S-adenosylmethionine (27). An activation system exists in cellular extracts of A. dehalogenans that can activate the veratrol:tetrahydrofolate three-component system and catalyze the direct reduction of the veratrol-specific corrinoid protein to the Co(I) state; however, the activating protein has not been purified (31).For the methanogen methylamine and methanol methyltransferase systems, an activation process is readily detectable in cell extracts that is ATP- and hydrogen-dependent (32, 33). Daas et al. (34, 35) examined the activation of the methanol methyltransferase system in M. barkeri and purified in low yield a methyltransferase activation protein (MAP) which in the presence of a preparation of hydrogenase and uncharacterized proteins was required for ATP-dependent reductive activation of methanol:CoM methyl transfer. MAP was found to be a heterodimeric protein without a UV-visible detectable prosthetic group. Unfortunately, no protein sequence has been reported for MAP, leaving the identity of the gene in question. The same MAP protein was also suggested to activate methylamine:CoM methyl transfer, but this suggestion was based on results with crude protein fractions containing many cellular proteins other than MAP (36).Here we report of the identification and purification to near-homogeneity of RamA (reductive activation of methyltransfer, amines), a protein mediating activation of methylamine:CoM methyl transfer in a highly purified system (Fig. 1). Quite unlike MAP, which was reported to lack prosthetic groups, RamA is an iron-sulfur protein that can catalyze reduction of a corrinoid protein such as MtmC to the Co(I) state in an ATP-dependent reaction (Fig. 1). Peptide mapping of RamA allowed identification of the gene encoding RamA and its homologs in the genomes of Methanosarcina spp. RamA belongs to COG3894, a group of uncharacterized metal-binding proteins found in a number of genomes. RamA, thus, provides a functional example for a family of proteins widespread among bacteria and Archaea whose physiological role had been largely unknown.     

Characterization of an O-demethylase of Desulfitobacterium hafniense DCB-2

Besides acetogenic bacteria, only Desulfitobacterium has been described to utilize and cleave phenyl methyl ethers under anoxic conditions; however, no ether-cleaving O-demethylases from the latter organisms have been identified and investigated so far. In this study, genes of an operon encoding O-demethylase components of Desulfitobacterium hafniense strain DCB-2 were cloned and heterologously expressed in Escherichia coli. Methyltransferases I and II were characterized. Methyltransferase I mediated the ether cleavage and the transfer of the methyl group to the superreduced corrinoid of a corrinoid protein. Desulfitobacterium methyltransferase I had 66% identity (80% similarity) to that of the vanillate-demethylating methyltransferase I (OdmB) of Acetobacterium dehalogenans. The substrate spectrum was also similar to that of the latter enzyme; however, Desulfitobacterium methyltransferase I showed a higher level of activity for guaiacol and used methyl chloride as a substrate. Methyltransferase II catalyzed the transfer of the methyl group from the methylated corrinoid protein to tetrahydrofolate. It also showed a high identity (～70%) to methyltransferases II of A. dehalogenans. The corrinoid protein was produced in E. coli as cofactor-free apoprotein that could be reconstituted with hydroxocobalamin or methylcobalamin to function in the methyltransferase I and II assays. Six COG3894 proteins, which were assumed to function as activating enzymes mediating the reduction of the corrinoid protein after an inadvertent oxidation of the corrinoid cofactor, were studied with respect to their abilities to reduce the recombinant reconstituted corrinoid protein. Of these six proteins, only one was found to catalyze the reduction of the corrinoid protein.     

Role of the fused corrinoid/methyl transfer protein CmtA during CO-dependent growth of Methanosarcina acetivorans

The genome of Methanosarcina acetivorans encodes three homologs, initially annotated as hypothetical fused corrinoid/methyl transfer proteins, which are highly elevated in CO-grown cells versus cells grown with alternate substrates. Based only on phenotypic analyses of deletion mutants, it was previously concluded that the homologs are strictly dimethylsulfide:coenzyme M (CoM) methyltransferases not involved in the metabolism of CO (E. Oelgeschlager and M. Rother, Mol. Microbiol. 72:1260 -1272, 2009). The homolog encoded by MA4383 (here designated CmtA) was reexamined via biochemical characterization of the protein overproduced in Escherichia coli. Purified CmtA reconstituted with methylcob(III)alamin contained a molar ratio of cobalt to protein of 1.0 ± 0.2. The UV-visible spectrum was typical of methylated corrinoid-containing proteins, with absorbance maxima at 370 and 420 nm and a band of broad absorbance between 450 and 600 nm with maxima at 525, 490, and 550 nm. CmtA reconstituted with aquocobalamin showed methyl-tetrahydromethanopterin:CoM (CH(3)-THMPT:HS-CoM) methyltransferase activity (0.31 μmol/min/mg) with apparent K(m) values of 135 μM for CH(3)-THMPT and 277 μM for HS-CoM. The ratio of CH(3)-THMPT:HS-CoM methyltransferase activity in the soluble versus membrane cellular fractions was 15-fold greater in CO-grown versus methanol-grown cells. A mutant strain deleted for the CmtA gene showed lower growth rates and final yields when cultured with growth-limiting partial pressures of CO, demonstrating a role for CmtA during growth with this substrate. The results establish that CmtA is a soluble CH(3)-THSPT:HS-CoM methyltransferase postulated to supplement the membrane-bound CH(3)-THMPT:HS-CoM methyltransferase during CO-dependent growth of M. acetivorans. Thus, we propose that the name of the enzyme encoded by MA4384 be CmtA (for cytoplasmic methyltransferase).     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997