Electron paramagnetic resonance spectroscopic and electrochemical characterization of the partially purified N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanosarcina mazei Gö1

Lu, Weiping; Becher, B; Gottschalk, Gerhard; Ragsdale, Stephen W.

doi:10.1128/jb.177.9.2245-2250.1995

Cited by 21 publications

(22 citation statements)

References 33 publications

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“…Here, the observed midpoint redox potentials about equaled the E 0 Ј ϭ Ϫ500 mV of the free base-off cob(II)-amide/cob(I)amide couple. For a number of corrinoid-containing methyltransferases, midpoint potentials have been measured that were significantly higher than found for the corresponding B 12 derivatives in solution (25)(26)(27)(28). For example, the reduction of base-on cob(II)amide to cob(I)amide in the membrane-bound methyltetrahydromethanopterin:HS-CoM methyltransferase complex from Methanosarcina mazei…”

Section: Discussionmentioning

confidence: 98%

Activation Mechanism of Methanol:5-Hydroxybenzimidazolylcobamide Methyltransferase from

Daas

Hagen

Keltjens

et al. 1996

Journal of Biological Chemistry

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Methanosarcina barkeri can utilize methanol as sole source for methanogenesis and growth. The first step in the reduction of methanol to methane is the formation of an enzyme-bound methylcobamide catalyzed by methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT 1 ) 1 (1). The methyl group of methylated MT 1 is subsequently transferred to 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) by Co-methyl-5-hydroxybenzimidazolylcobamide:HS-CoM methyltransferase (MT 2 ) (2). As a result methyl-coenzyme M (CH 3 -S-CoM) is produced, which is the substrate for the final step in methanogenesis in all methanogens studied so far (3).The corrinoid prosthetic group of MT 1 can only be methylated by methanol when the central cobalt atom of the cobamide is present in the highly reduced Co(I) state (4, 5). Since this state is extremely sensitive toward oxidation, MT 1 readily inactivates upon manipulation and even during catalysis. Reactivation is possible and requires participation of a reducing system, methyltransferase activation protein (MAP), and ATP (4, 6 -8). The reducing system consists of hydrogen, hydrogenase, and ferredoxin. Ferredoxin is not absolutely required, though it stimulates the apparent reaction rate of methyl group transfer (6).Here, we report the UV-visible absorbance and electron paramagnetic resonance properties of the corrinoid prosthetic groups of MT 1 under various additions of the reducing system, MAP, and ATP. From these results, the sequence of events leading to the formation of the cob(I)amide of MT 1 is deduced. The activation of MT 1 proceeds by a novel mechanism, which is presented in a model and compared to those described for other corrinoid-containing methyl group-transferring proteins. EXPERIMENTAL PROCEDURESCell Material-Cells of M. barkeri strain MS (DSM 800) were grown and harvested, and cell extract was prepared anaerobically as described previously (6, 9).Enzyme Assays-Incubation mixtures were prepared in an anaerobic glove box, and reactions were performed in crimp-sealed 10-ml serum vials. MT 1 activity was determined by measuring the methanol-dependent HS-CoM conversion to CH 3 -S-CoM when added to a reaction mixture containing MT 2 /hydrogenase, MAP, and ferredoxin fractions obtained after DEAE-Sepharose fractionation of cell extract of M. barkeri (6). A typical reaction mixture (final volume, 100 l) contained 50 mM TES/K ϩ buffer (pH 7.0), 24 mM MgCl 2 , 10 mM methanol, 10 mM HSCoM, 2 mM ATP, 1 mM 2-bromoethanesulfonic acid, 20 l of MT 2 / hydrogenase fraction, 5 l of ferredoxin fraction, 25 l of MAP fraction, and an amount (usually 25 l) of MT 1 to be tested (6). After gassing with 50% H 2 , 50% N 2 (100 kPa), the vials were kept on ice. Reactions were started by placing the vials at 37°C. After appropriate incubation periods, routinely 0, 15, 30, 45, and 60 min, reactions were stopped by placing the vials on ice. Activity of methyl group transfer of methanol to HS-CoM was routinely assayed by measuring the decrease in the amount of HS-CoM (see below). The methyltransfe...

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Section: Discussionmentioning

confidence: 98%

Activation Mechanism of Methanol:5-Hydroxybenzimidazolylcobamide Methyltransferase from

Daas

Hagen

Keltjens

et al. 1996

Journal of Biological Chemistry

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“…This is consistent with the involvement of the corrinoid bound to the ␤ subunit. Corrinoid-dependent methyltransferases are in the Co(I) state following methyl group transfer (1,2,16,19,21,25,33). In this strongly nucleophilic, but oxygen-sensitive, state, they can be remethylated by the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…Transmethylation is apparently coupled to the translocation of sodium ions across the cytoplasmic membrane, thus forming a site of chemiosmotic energy conservation in methanogens (3). The protein has been purified from Methanosarcina mazei Gö1 (33) and M. thermoautotrophicum (15,27), with variable numbers of subunits. Eight genes have been identified in the transcriptional unit encoding the methyltransferase in the latter organism (23,39).…”

mentioning

confidence: 99%

Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri

Tallant

Krzycki

1996

J Bacteriol

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“…Four different enzymes capable of methylating CoM have been identified (19,29,36,55,58,60). All four CoM methylases either bind corrinoid or interact with proteins binding a corrinoid cofactor.…”

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confidence: 99%

Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase

Paul

Krzycki

1996

J Bacteriol

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The sequence and transcript of the genes encoding a recently discovered coenzyme M methylase in Methanosarcina barkeri were analyzed. This 480-kDa protein is composed of two subunits in equimolar concentrations which bind one corrinoid cofactor per ␣␤ dimer. The gene for the ␣ polypeptide, mtsA, is upstream of that encoding the ␤ polypeptide, mtsB. The two genes are contiguous and overlap by several nucleotides. A 1.9-kb mRNA species which reacted with probes specific for either mtsA or mtsB was detected. Three possible methanogen consensus BoxA sequences as well as two sets of direct repeats were found upstream of mtsA. The 5 end of the mts transcript was 19 nucleotides upstream of the translational start site of mtsA and was positioned 25 bp from the center of the proximal BoxA sequence. The transcript was most abundant in cells grown to the late log phase on acetate but barely detectable in cells grown on methanol or trimethylamine. The amino acid sequence of MtsB was homologous to the cobalamin-binding fragment of methionine synthase from Escherichia coli and possessed the signature residues involved in binding the corrinoid, including a histidyl residue which ligates cobalt. The sequence of MtsA is homologous to the "A" and "M" isozymes of methylcobamide:coenzyme M methyltransferases (methyltransferase II), indicating that the ␣ polypeptide is a new member of the methyltransferase II family of coenzyme M methylases. All three methyltransferase II homolog sequences could be aligned with the sequences of uroporphyrinogen decarboxylase from various sources. The implications of these homologies for the mechanism of corrinoid binding by proteins involved in methylotrophic methanogenesis are discussed.

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Electron paramagnetic resonance spectroscopic and electrochemical characterization of the partially purified N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanosarcina mazei Gö1

Cited by 21 publications

References 33 publications

Activation Mechanism of Methanol:5-Hydroxybenzimidazolylcobamide Methyltransferase from

Activation Mechanism of Methanol:5-Hydroxybenzimidazolylcobamide Methyltransferase from

Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri

Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase

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