Abstract
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.
Highlights
Methanosarcina species and related genera comprise a branch of methanogenic euryarcheaota capable of methylotrophic methanogenesis from compounds such as methanol, trimethylamine (TMA),1 dimethylamine (DMA), and monometh
Heterologous Expression of MtsA—Methylcobalamin:coenzyme M (CoM) methyltransferase activity was quantified in cells of E. coli BL21(DE3)pLysS transformed with pET-40ex using an assay based on cyanide derivatization of the hydroxycobalamin produced from oxidation of the cob(I)alamin generated by demethylation of methylcobalamin [42]
Preparations of recombinant MtsA made without addition of zinc typically had 3-fold lower methylcobalamin:CoM methyltransferase activity than MtsA refolded in the presence of added zinc
Summary
Methanosarcina species and related genera comprise a branch of methanogenic euryarcheaota capable of methylotrophic methanogenesis from compounds such as methanol, trimethylamine (TMA),1 dimethylamine (DMA), and monometh-. MtsA in association with MtsB (the native methylthiol:CoM methyltransferase) catalyzed methylcobalamin:CoM methyl transfer at 0.4 mol minϪ1 mgϪ1 [34]. Recombinant MtsA catalyzes methylcobalamin:CoM methyl transfer activity, confirming the previously reported role of this subunit in the demethylation of corrinoid bound to MtsB and methylation of CoM.
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