Open AccessMethanol : coenzyme M methyltransferase from Methanosarcina barkeri
Author(s) -
Sauer Karin,
Thauer Rudolf K.
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2530698.x
Subject(s) - corrinoid , methanosarcina barkeri , heterologous expression , cofactor , chemistry , biochemistry , escherichia coli , histidine , mutagenesis , site directed mutagenesis , enzyme , biology , stereochemistry , methyltransferase , recombinant dna , dna , organic chemistry , methane , methylation , methanogenesis , mutant , mutation , gene
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 methylcob(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 Co 2+ 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.