Purification and characterization of dimethylamine:: 5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro

Dimethylamine: 5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent K-m = 0.45 mM) and 7-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (K-i = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B-12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein. ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.