Reconstitution of dimethylamine : coenzyme M methyl transfer with a discrete corrinoid protein and two methyltransferases purified from Methanosarcina barkeri

Methyl transfer from dimethylamine to coenzyme M was reconstituted in vitro for the first time using only highly purified proteins. These proteins iso lated from Methanosarcina barkeri included the previously unidentified corr inoid protein MtbC, which copurified with MtbA, the methylcorrinoid:Coenzym e M methyltransferase specific for methanogenesis from methylamines, MtbC b inds 1.0 mel of corrinoid cofactor/mol of 24-kDa polypeptide and stimulated dimethylamine:coenzyme M methyl transfer 3.4-fold in a cell extract. Purif ied MtbC and MtbA were used to assay and purify a dimethylamine:corrinoid m ethyltransferase, MtbB1. MtbB1 is a 230-kDa protein composed of 51-kDa subu nits that do not possess a corrinoid prosthetic group. Purified MtbB1, MtbC , and MtbA were the sole protein requirements for in vitro dimethylamine:co enzyme M methyl transfer. An MtbB1:MtbC ratio of 1 was optimal for coenzyme M methylation with dimethylamine, MtbB1 methylated either corrinoid bound to MtbC or free co-b(I)alamin with dimethylamine, indicating MtbB1 carries an active site for dimethylamine demethylation and corrinoid methylation. E xperiments in which different proteins of the resolved monomethylamine:coen zyme M methyl transfer reaction replaced proteins involved in dimethylamine :coenzyme M methyl transfer indicated high specificity of MtbB1 and MtbC in dimethylamine: coenzyme M methyl transfer activity. These results indicate MtbB1 demethylates dimethylamine and specifically methylates the corrinoid prosthetic group of MtbC, which is subsequently demethylated by MtbA to me thylate coenzyme M during methanogenesis from dimethylamine.