SPECIFIC ROLES OF METHYLCOBAMIDE-COENZYME-M METHYLTRANSFERASE ISOZYMES IN METABOLISM OF METHANOL AND METHYLAMINES IN METHANOSARCINA-BARKERI

An immunochemical approach was employed as a direct test for functiona l activities of isozymes of methylcobamide:coenzyme M methyltransferas e (MT2-M and MT2-A) in the metabolic pathways of methane formation fro m: methanol, acetate, monomethylamine, dimethylamine, and trimethylami ne. Specific removal of the MT2 isozymes from buffer soluble cell extr acts of Methano-sarcina barkeri was accomplished by use of immobilized , affinity-purified, ovine polyclonal antibodies. Extracts of methanol -grown cells depleted of MT2-M lost entirely the ability to carry out conversion of methanol to 2-(methylthio)ethanesulfonate (methyl-CoM). Methanol:CoM methyl transfer activity was completely restored by addit ion of purified MT2-M, but no activity was recovered by addition of MT 2-A In contrast, the activity of trimethylamine-grown cell extracts to convert monomethylamine and dimethylamine to methyl-CoM was lost almo st entirely by immunosorptive removal of MT2-A. Addition of purified M T2-A, but not MT2-M, to the MT2-A-depleted extract fully reconstituted methyl-CoM formation from both mono- and dimethylamine. Interestingly , in extracts resolved of MT2-A, trimethylamine-dependent methylation of coenzyme M was observed at approximately 20% of the rate of control s not treated with antibody. Furthermore, both isozymes were effective in full restoration of trimethylamine conversion. Tests indicated tha t neither of the two MT2 isozymes are involved in methane formation fr om acetate. The results establish that MT2-A plays a specific role in metabolism of methylated amine substrates, whereas, MT2-M functions in methane formation from trimethylamine and methanol.