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1.
Methyl-coenzyme M formation from coenzyme M and methanol in Methanosarcina barkeri is catalysed by an enzyme system composed of three polypeptides MtaA, MtaB and MtaC, the latter of which harbours a corrinoid prosthetic group. We report here that MtaC can be substituted by free cob(I)alamin which is methylated with methanol in an MtaB-catalysed reaction and demethylated with coenzyme M in an MtaA-catalysed reaction. Methyl transfer from methanol to coenzyme M was found to proceed at a relatively high specific activity at micromolar concentrations of cob(I)alamin. This finding was surprising because the methylation of cob(I)alamin catalysed by MtaB alone and the demethylation of methylcob(III)alamin catalysed by MtaA alone exhibit apparent Km for cob(I)alamin and methylcob(III)alamin of above 1 mm. A possible explanation is that MtaA positively affects the MtaB catalytic efficiency and vice versa by decreasing the apparent Km for their corrinoid substrates. Activation of MtaA by MtaB was methanol-dependent. In the assay for methanol:coenzyme M methyltransferase activity cob(I)alamin could be substituted by cob(I)inamide which is devoid of the nucleotide loop. Substitution was, however, only possible when the assays were supplemented with imidazole: approximately 1 mm imidazole being required for half-maximal activity. Methylation of cob(I)inamide with methanol was found to be dependent on imidazole but not on the demethylation of methylcob(III)inamide with coenzyme M. The demethylation reaction was even inhibited by imidazole. The structure and catalytic mechanism of the MtaABC complex are compared with the cobalamin-dependent methionine synthase. 相似文献
2.
Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes. 相似文献
3.
In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex
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Ming Dong Tara D. Gonzalez Meghan M. Klems Lisa M. Steinberg Wilfred Chen Eleftherios T. Papoutsakis Brian J. Bahnson 《Biotechnology progress》2017,33(5):1243-1249
Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel‐precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243–1249, 2017 相似文献
4.
Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri. 总被引:1,自引:0,他引:1
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Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa corrinoid protein previously identified by its methylation following inhibition of methyl-CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha subunit is responsible for the activity. The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated corrinoid bound to the beta subunit of the 480-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high-pressure liquid chromatography with indirect photometric detection. Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cofactor binding subunit during isolation. 相似文献
5.
Markus Krüer Michael Haumann Wolfram Meyer-Klaucke Rudolf K Thauer Holger Dau 《European journal of biochemistry》2002,269(8):2117-2123
Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237-->Ala and Cys239-->Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding. 相似文献
6.
The Moorella thermoacetica aromatic O-demethylase was characterized as an inducible three-component system with similarity to the methanogenic methanol, methylamine, and methanethiol methyltransferases and to the O-demethylase system from Acetobacterium dehalogenans. MtvB catalyzes methyl transfer from a phenylmethylether to the cobalt center of MtvC, a corrinoid protein. MtvA catalyzes transmethylation from MtvC to tetrahydrofolate, forming methyltetrahydrofolate. Cobalamin can substitute for MtvC. 相似文献
7.
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). 相似文献
8.
Ferguson DJ Gorlatova N Grahame DA Krzycki JA 《The Journal of biological chemistry》2000,275(37):29053-29060
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. 相似文献
9.
Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro. 总被引:1,自引:0,他引:1
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R W Wassenaar P J Daas W J Geerts J T Keltjens C van der Drift 《Journal of bacteriology》1996,178(23):6937-6944
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. 相似文献
10.
A Studer E Stupperich S Vuilleumier T Leisinger 《European journal of biochemistry》2001,268(10):2931-2938
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. 相似文献
11.
Involvement of an activation protein in the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri. 总被引:3,自引:2,他引:1
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P J Daas K A Gerrits J T Keltjens C van der Drift G D Vogels 《Journal of bacteriology》1993,175(5):1278-1283
Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes required for transfer of the methyl group of methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 binds the methyl group of methanol to its corrinoid prosthetic group only when the central cobalt atom of the corrinoid is present in the highly reduced Co(I) state. However, upon manipulation of MT1 and even during catalysis, the enzyme becomes inactivated as the result of Co(I) oxidation. Reactivation requires H2, hydrogenase, and ATP. Ferredoxin stimulated the apparent reaction rate of methyl group transfer. Here we report that one more protein fraction was found essential for the overall reaction and, more specifically, for formation of the methylated MT1 intermediate. The more of the protein that was present, the shorter the delay of the start of methyl group transfer. The maximum velocity of methyl transfer was not substantially affected by these varying amounts of protein. This demonstrated that the protein was involved in the activation of MT1. Therefore, it was called methyltransferase activation protein. 相似文献
12.
Methyl-coenzyme M (2-methylthioethane sulfonate) is the key intermediate of methane formation in methanogenic archaea. It is generated from coenzyme M (2-mercaptoethane sulfonate) in methyl transfer reactions catalyzed by proteins containing zinc. Here, we report that, for methyltransferase MtaA from Methanosarcina barkeri, the zinc is involved in coenzyme M activation. For the experiments an inactive MtaA apoprotein was obtained by heterologous overproduction in Escherichia coli grown in the presence of 2 mM EDTA. The apoprotein was found to react with zinc or cobalt to the fully active holoenzyme. Appoximately 1 mol of transition metal was bound per mol of protein. Upon incubation of the holoenzyme with coenzyme M approximately 1 mol of proton was released per mol of zinc or cobalt. Protons were not released upon incubation of the apoprotein with coenzyme M or of the holoprotein with other thiol compounds or with methyl-coenzyme M. The findings are interpreted as indicating that the role of the transition metal in MtaA is to lower the microscopic pKa of the thiol group of coenzyme M by coordination to the zinc, and thus to increase its nucleophilicity for methyl group attack. The pKZn2+ of MtaA was re-determined and found to be > 15 and not 9.6 as previously reported by us. 相似文献
13.
McAnulla C Woodall CA McDonald IR Studer A Vuilleumier S Leisinger T Murrell JC 《Applied and environmental microbiology》2001,67(1):307-316
Hyphomicrobium chloromethanicum CM2(T), an aerobic methylotrophic member of the alpha subclass of the class proteobacteria, can grow with chloromethane as the sole carbon and energy source. H. chloromethanicum possesses an inducible enzyme system for utilization of chloromethane, in which two polypeptides (67-kDa CmuA and 35-kDa CmuB) are expressed. Previously, four genes, cmuA, cmuB, cmuC, and purU, were shown to be essential for growth of Methylobacterium chloromethanicum on chloromethane. The cmuA and cmuB genes were used as probes to identify homologs in H. chloromethanicum. A cmu gene cluster (9.5 kb) in H. chloromethanicum contained 10 open reading frames: folD (partial), pduX, orf153, orf207, orf225, cmuB, cmuC, cmuA, fmdB, and paaE (partial). CmuA from H. chloromethanicum (67 kDa) showed high identity to CmuA from M. chloromethanicum and contains an N-terminal methyltransferase domain and a C-terminal corrinoid-binding domain. CmuB from H. chloromethanicum is related to a family of methyl transfer proteins and to the CmuB methyltransferase from M. chloromethanicum. CmuC from H. chloromethanicum shows identity to CmuC from M. chloromethanicum and is a putative methyltransferase. folD codes for a methylene-tetrahydrofolate cyclohydrolase, which may be involved in the C(1) transfer pathway for carbon assimilation and CO(2) production, and paaE codes for a putative redox active protein. Molecular analyses and some preliminary biochemical data indicated that the chloromethane utilization pathway in H. chloromethanicum is similar to the corrinoid-dependent methyl transfer system in M. chloromethanicum. PCR primers were developed for successful amplification of cmuA genes from newly isolated chloromethane utilizers and enrichment cultures. 相似文献
14.
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. 相似文献
15.
Methylthiol:coenzyme M methyltransferase from Methanosarcina barkeri, an enzyme of methanogenesis from dimethylsulfide and methylmercaptopropionate. 总被引:1,自引:0,他引:1
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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. 相似文献
16.
Tsuneo Ferguson Jitesh A. Soares Tanja Lienard Gerhard Gottschalk Joseph A. Krzycki 《The Journal of biological chemistry》2009,284(4):2285-2295
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 Em 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. 相似文献
17.
18.
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. 相似文献
19.
Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri. 总被引:4,自引:0,他引:4
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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. 相似文献
20.
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. 相似文献