Methanol: Coenzyme M Methyltransferase from Methanosarcina Barkeri

Abstract

In Methaizosarcina burkeri, 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 MT 1 and MT2. Transferase MTI 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) ELK J. Riochem. 235, 653-6591, We report here on the corresponding analysis of MTl. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 pmol min-' mg-'. The enzyme catalyzed the methylation of its bound corrinoid in the cob(1)amide oxidation state by methanol. In addition to this autoinethylation, the purified enzyme was found to catalyze the methylation of free cob(1)alamin to methylcob(II1)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 mtuC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtuCB. The corrinoid-harbouring subunit MtaC exhibits 35 % sequence similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli.

Insight into the mechanism of biological methanol activation based on the crystal structure of the methanol-cobalamin methyltransferase complex

“…The tight binding of the corrinoid even in the tetra-coordinated cob(I) oxidation state was confirmed for MtaC by biochemical studies (6). Interestingly, the corrinoid rings significantly differ in their conformations because of their loose contact to the Rossmann domain and their distinctive interactions with segments of other subunits approaching the ␤-face.…”

“…MtaB catalyzes the methylation of the MtaC-bound cob(I)amide (reaction 2), and MtaA catalyzes the transfer of the methyl group from the MtaC-bound methylcob(III)amide to coenzyme M (reaction 3). In the two reactions MtaC can be substituted by free cob(I)alamin and free methylcob(III)alamin, respectively (4,(6)(7)(8)(9)(10).…”

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

“…However, it remains to be shown if this regulation is dose-dependent for the whole Methanosarcina population, as is the case for tetO/TetR systems in bacteria and eukaryotes, or an autocatalytic induction of expression due to active uptake of the inducer, as is the case for Plac-and Para-dependent gene expression (Novick andWeiner 1957, Morgan-Kiss et al 2002). With the Pmcr(tetO)/TetR system established for Methanosarcina it seems feasible now to overproduce enzymes in a catalytically active form where other host/overexpresion systems have resulted in partially inactive protein (Roberts et al 1989, Sauer et al 1997, Sauer and Thauer Rudolf 1998, Loke et al 2000. Furthermore, even toxic genes can probably be overproduced because of the tight repression of the hybrid promoter in the absence of tetracycline.…”

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

“…The sequence of genes encoding the polypeptides effecting CoM methylation with methylamines (26, 30 -32) and methanol (24,32) have revealed similarities to the two subunits of the methylthiol:CoM methyltransferase (33) (see Fig. 1 and accompanying legend).…”

“…In the case of the methylamines, the methylcorrinoid:CoM methyltransferase is a single protein, MtbA (29). A different yet homologous protein, MtaA, methylates CoM with the methanol cognate corrinoid protein, MtaC (24).…”