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Abstract

The enzyme system catalyzing the formation of methyl-coenzyme M from
methanol and coenzyme M in Methanosarcina barkeri is composed of the
three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a
corrinoid prosthetic group. The heterologous expression of mtaA and mtaB
in Escherichia coli has been described previously. We report here on the
overproduction of the apoprotein of MtaC in E. coli, on its
reconstitution to the active holoprotein with either cob(II)alamin or
methyl-cob(III)alamin, and on the properties of the reconstituted
corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound
cobamide/mol. EPR spectroscopic evidence is presented for a His residue
as an axial ligand to Co2+ of the bound corrinoid. This active-site His
was identified by site-directed mutagenesis as His136 in the MtaC
sequence that contains four His residues. The reconstituted MtaC, in the
cob(I)amide oxidation state, was methylated with methanol in the
presence of MtaB and demethylated with coenzyme M in the presence of
MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was
formed from methanol and coenzyme M at specific rates comparable to
those determined for the enzyme system purified from M. barkeri. M.
barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme
reacted with MtaC with only 2.5 % of the activity of MtaA.