Publications

• Tetrahydromethanopterin methyltransferase, a component of the methane synthesizing complex of Methanobacterium thermoautotrophicum.

  Sauer F.D.

  A new enzyme, tetrahydromethanopterin methyltransferase, which catalyzes the transfer of methyl groups from methyl-tetrahydromethanopterin to 2-mercaptoethane-sulfonate, has been found in the methane synthesizing complex of Methanobacterium thermoautotrophicum. The enzyme is oxygen sensitive and h ... >> More

  A new enzyme, tetrahydromethanopterin methyltransferase, which catalyzes the transfer of methyl groups from methyl-tetrahydromethanopterin to 2-mercaptoethane-sulfonate, has been found in the methane synthesizing complex of Methanobacterium thermoautotrophicum. The enzyme is oxygen sensitive and has a well defined pH optimum at pH 6.7. There was no methyl group transfer when the enzyme was heated to 100 degrees for 5 min. The product of the forward reaction, methyl-CoM, was positively identified by TLC and high voltage paper electrophoresis. The demethylation of methyl-CoM, in the absence of methane synthesis, was dependent on the addition of H4MPT which suggests that the enzyme reaction is reversible. << Less

  Biochem Biophys Res Commun 136:542-547(1986) [PubMed] [EuropePMC]

• The energetics and sodium-ion dependence of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase studied with cob(I)alamin as methyl acceptor and methylcob(III)alamin as methyl donor.

  Weiss D.S., Gartner P., Thauer R.K.

  N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane-associated enzyme complex that uses a methyl-transfer reaction to drive an energy-conserving sodium-ion pump. Methyl transfer occurs in two steps, first from N5-methyltetrahydromethanopterin (CH3- ... >> More

  N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane-associated enzyme complex that uses a methyl-transfer reaction to drive an energy-conserving sodium-ion pump. Methyl transfer occurs in two steps, first from N5-methyltetrahydromethanopterin (CH3-H4MPT) to an enzyme-bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H-S-CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH3-H4MPT and cob(I)alamin (reaction a), and methyl-coenzyme M formation from methylcob(III)alamin and H-S-CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH3-H4MPT to H-S-CoM. The free energy changes (delta G0') associated with reactions a and b were both between -10 kJ/mol and -20 kJ/mol, consistent with a free energy change of approximately -30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium-ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme-bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H-S-CoM is a site of coupling with sodium-ion translocation. << Less

  Eur J Biochem 226:799-809(1994) [PubMed] [EuropePMC]

• The energy conserving N5-methyltetrahydromethanopterin:coenzyme M methyltransferase complex from Methanobacterium thermoautotrophicum is composed of eight different subunits.

  Harms U., Weiss D.S., Gaertner P., Linder D., Thauer R.K.

  N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum strain Marburg is a membrane-associated enzyme complex which catalyzes an energy-conserving, sodium-ion-translocating step in methanogenesis from H2 and CO2. We report here that the comple ... >> More

  N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum strain Marburg is a membrane-associated enzyme complex which catalyzes an energy-conserving, sodium-ion-translocating step in methanogenesis from H2 and CO2. We report here that the complex is composed of eight different subunits for which evidence was obtained at the protein, DNA and RNA levels: (a) SDS/PAGE of the purified complex revealed the presence of eight different polypeptides of apparent molecular masses of 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 (MtrD), 13 (MtrG), 12.5 (MtrB) and 12 kDa (MtrF). The N-terminal amino acid sequences of the 12-, 12.5- and 13-kDa polypeptides, which had previously not been accessible, were determined; (b) cloning and sequencing of the corresponding genes revealed the presence of the eight mtr genes organized in a 4.9-kbp gene cluster in the order mtrEDCBAFGH; (c) Northern-blot analysis revealed the presence of a 5-kbp transcript. DNA probes derived from the mtrE and mtrH genes hybridized to the transcript, indicating that the eight mtr genes are organized in a transcription unit. By primer extension, the 5' end of the mtrEDC-BAFGH mRNA was analyzed. The mtr operon was found to be located between the methyl-coenzyme M reductase I operon (mcr) and a downstream open reading frame predicted to encode a Na+/Ca2+, K+ exchanger. << Less

  Eur. J. Biochem. 228:640-648(1995) [PubMed] [EuropePMC]

• Purification and properties of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanobacterium thermoautotrophicum.

  Gaertner P., Ecker A., Fischer R., Linder D., Fuchs G., Thauer R.K.

  N5-Methyltetrahydromethanopterin:coenzyme M meth-yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy-conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purifi ... >> More

  N5-Methyltetrahydromethanopterin:coenzyme M meth-yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy-conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30-fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native 670-kDa enzyme was found to contain 7.6 mol 5-hydroxybenzimidazolyl cobamide/mol, 37 mol non-heme iron/mol and 34 mol acid-labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23-kDa polypeptide. The apparent molecular masses of the polypeptides given above were determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usually done, the 23-kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21-kDa, 24-kDa, and 28-kDa polypeptides formed aggregates. The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 mumol.min-1.mg protein-1. The apparent Km for N5-methyltetrahydromethanopterin was 260 microM and that for coenzyme M was 60 microM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5-2-fold. << Less

  Eur. J. Biochem. 213:537-545(1993) [PubMed] [EuropePMC]

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

  Vepachedu V.R., Ferry J.G.

  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 pre ... >> More

  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). << Less

  J Bacteriol 194:4161-4168(2012) [PubMed] [EuropePMC]

• The Na(+)-translocating methyltransferase complex from methanogenic archaea.

  Gottschalk G., Thauer R.K.

  Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-asso ... >> More

  Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N(5)-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (DeltaG degrees '=-30 kJ/mol) is catalyzed by a Na(+)-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA-H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions. << Less

  Biochim Biophys Acta 1505:28-36(2001) [PubMed] [EuropePMC]