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

Abstract

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.

The MtsA Subunit of the Methylthiol:Coenzyme M Methyltransferase of Methanosarcina barkeri Catalyses Both Half-reactions of Corrinoid-dependent Dimethylsulfide: Coenzyme M Methyl Transfer

“…MtsB is highly similar to the entire length of methylotrophic corrinoid proteins such as MttC, MtbC, or MtmC (30,33). These latter proteins bind one corrinoid cofactor per polypeptide (21,26,27,35).…”

“…MtsA shares ϳ50% similarity over nearly its entire length to the CoM methylases involved in CoM methylation by methanol (MtaA) and methylamine (MtbA) (33), and, as one would predict, they share some similarities. Like MtaA and MtbA, MtsA catalyzes an active methylcobalamin:CoM methyl transfer reaction.…”

“…Like MtaA and MtbA, MtsA catalyzes an active methylcobalamin:CoM methyl transfer reaction. The sequence of all three proteins (32,33) possess the proposed binding motif (V/I)LHICG, where zinc may coordinate to the His and Cys residues, as well as to a second Cys residue found 75 residues C-terminal to the first Cys residue (32,46). MtsA, like MtbA and MtaA, binds 1 mol of zinc/mol of polypeptide.…”

“…The methylamine cognate corrinoid proteins each bind one corrinoid per polypeptide (21,26,35), and these proteins as a group are homologous to the cobalamin binding domains of methionine synthase, as well as coenzyme B 12 -dependent enzymes (30,33,36). MtsB contains all expected signature residues for binding corrinoid (33,37) and is therefore the likely corrinoid binding subunit of methylthiol: CoM methyltransferase. Thus, a clear route for the demethylation of methylated MtsB corrinoid protein and methylation of CoM can be proposed, in that MtsA mediates the demethylation of MtsB and methylation of CoM (Fig.…”

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

“…were found, but these were not near corrinoid proteins or their associated methyltransferases. An exception was a gene encoding a Ram-like homolog found in M. barkeri Fusaro (Mbar_A3583) near the genes most similar to the methylthiol:CoM methyltransferase, a corrinoid-dependent methyltransferase homologous to methylamine corrinoid proteins and methylcobamide:CoM methyltransferases (47,48). A RamA homolog (MA0849) was also found in M. acetivorans adjacent to a gene encoding a methylcobamide:CoM methyltransferase homolog.…”