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The energy-conserving corrinoid-containing MtrA-H complex from Methanobacterium thermoautotrophicum is composed of eight different subunits of which MtrA harbors the corrinoid prosthetic group. EPR spectroscopic evidence has recently been presented for a histidine residue as a cobalt ligand of the cobamide [Harms, U. & Thauer, R. K. (1996a) Eur. J. Biochem. 241, 149-154]. This active site histidine was now identified by site-directed mutagenesis to be His84 in the MtrA sequence that contains three histidines. This result was substantiated by sequence comparison of MtrA from M. thermoautotrophicum, Methanococcus jannaschii, and Methanopyrus kandleri and of MtxA from Methanosarcina harkeri showing that only His84 is conserved. For comparison, the DNA sequences of the mtrEDCBAGH operon in M. kandleri and of the mtxXAH operon in M. barkeri were determined.

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methylcoenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated MT1 and MT2. Transferase MT1 is a corrinoid protein. The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously [Harms, U. and Thauer, R.K. (1996) Eur. J. Biochem. 235, 653-659]. We report here on the corresponding analysis of MT1. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 mumol min-1 mg-1. The enzyme catalyzed the methylation of its bound corrinoid in the cob(I)amide oxidation state by methanol. In addition to this automethylation, the purified enzyme was found to catalyze the methylation of free cob(I)alamin to methylcob(III)alamin. It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively. The subunit MtaC was shown to harbour the corrinoid prosthetic group. The genes mtaB and mtaC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtaCB. The corrinoid-harbouring subunit MtaC exhibits 35% sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum is a membrane-associated enzyme complex that catalyzes an energy-conserving, sodium ion translocating step in methanogenesis from H2 and CO2. The complex is composed of eight different subunits, MtrA-H, one of which (MtrA) harbours a corrinoid as prosthetic group. In this study, we report the structural properties of MtrA1 [des-(214-239)-MtrA], which is a deletion mutant of MtrA that lacks the last 25 C-terminal hydrophobic amino acids rendering the membrane protein soluble: (a) mtrA1 was heterologously expressed in Escherichia coli. Overexpression yielded a cytoplasmic protein which was purified approximately tenfold to apparent homogeneity. The purified protein was devoid of its corrinoid prosthetic group and not correctly folded as was evident from its electrophoretic mobility in SDS/PAGE. (b) Unfolding of MtrA1 with guanidine/HCl and refolding in the presence of cobalamin resulted in the formation of the correctly folded MtrA1 holoprotein that contained tightly bound cob(II)-alamin; the rate of reconstitution was highest when the refolding proceeded in the presence of titanium(III) citrate, which suggested that cob(I)alamin is the corrinoid species that binds to the apoprotein. (c) EPR spectra of the cob(II)alamin-containing holoprotein differentially labelled with 14N (nuclear spin 1) and 15N (nuclear spin 1/2) revealed that the corrinoid is bound to MtrA1 in the base-off form and that the Co(II) of the prosthetic group is coordinated by a histidine residue of the apoprotein. The results are interpreted with respect to the mechanism of energy conservation by the MtrA-H complex.

Methanosarcina barkeri is known to contain two methyltransferase isoenzymes, here designated MtaA and MtbA, which catalyze the formation of methyl-coenzyme M from methylcobalamin and coenzyme M. The genes encoding the two soluble 34-kDa proteins have been cloned and sequenced. mtaA and mtbA wee found to be located in different parts of the genome, each forming a monocystronic transcription unit. Northern blot analysis revealed that mtaA is preferentially transcribed when M. barkeri is grown on methanol and the mtbA gene when the organism is grown on H2/CO2 or trimethylamine. Comparison of the deduced amino acid sequences revealed the sequences of the two isoenzymes to be 37% identical. Both isoenzymes showed sequence similarity to uroporphyrinogen III decarboxylase from Escherichia coli. The mtaA gene was tagged with a sequence encoding six His placed bp before the mtaA start codon, and was functionally overexpressed in E. coli. 25% of the E. coli protein was found to be active methyltransferase which could be purified in two steps to apparent homogeneity with a 70% yield.

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.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane associated, corrinoid-containing enzyme complex which uses a methyl-transfer reaction to drive an energy-conserving sodium ion pump. The purified methyltransferase from Methanobacterium thermoautotrophicum (strain Marburg) exhibited a rhombic EPR signal indicative of a base-on cob(II)amide. In this form, the enzyme was almost completely inactive. Upon addition of Ti(III)citrate, which is a one-electron reductant known to reduce corrinoids to the cob(I)amide form, the EPR signal was completely quenched. In the reduced form, the enzyme was active. When the purified complex was incubated in the presence of both Ti(III) and N5-methyltetrahydromethanopterin (CH3-H4MPT), enzyme-bound Co-methyl-5'-hydroxybenzimidazolyl cob(III)amide was formed. Upon incubation of the methylated enzyme with either tetrahydromethanopterin or coenzyme M, the enzyme was demethylated with the concomitant formation of CH3-H4MPT and methylcoenzyme M, respectively. Enzyme demethylation, in contrast to enzyme methylation, was not dependent on the presence of Ti(III). Methyl transfer from the methylated enzyme to coenzyme M was essentially irreversible. These results are interpreted to that the purified enzyme complex is active only when the enzyme-bound corrinoid is in the reduced cob(I)amide form, and that methyl transfer from CH3-H4MPT to coenzyme M proceeds via nucleophilic attack of the cobalt(I) on the N5-methyl substituent of CH3-H4MPT, forming an enzyme-bound CH3-corrinoid as intermediate. Methyl-coenzyme M formation from CH3-H4MPT and coenzyme M, as catalyzed by the purified methyltransferase, was stimulated by sodium ions, half-maximal activity being obtained at approximately 50 microM Na+. We therefore infer that the methyltransferase, as isolated, is capable of vectorial sodium ion translocation.

Methanosarcina barkeri was recently shown to contain two cytoplasmic isoenzymes of methylcobalamin: coenzyme M methyltransferase (methyltransferase 2). Isoenzyme I predominated in methanol-grown cells and isoenzyme II in acetate-grown cells. It was therefore suggested that isoenzyme I functions in methanogenesis from methanol and isoenzyme II in methanogenesis from acetate. We report here that cells of M. barkeri grown on trimethylamine, H2/CO2, or acetate contain mainly isoenzyme II. These cells were found to have in common that they can catalyze the formation of methane from trimethylamine and H2, whereas only acetate-grown cells can mediate the formation of methane from acetate. Methanol-grown cells, which contained only low concentrations of isoenzyme II, were unable to mediate the formation of methane from both trimethylamine and acetate. These and other results suggest that isoenzyme II (i) is employed for methane formation from trimethylamine rather than from acetate, (ii) is constitutively expressed rather than trimethylamine-induced, and (iii) is repressed by methanol. The constitutive expression of isoenzyme II in acetate-grown M. barkeri can explain its presence in these cells. The N-terminal amino acid sequences of isoenzyme I and isoenzyme II were analyzed and found to be only 55% similar.