COENZYME-M METHYLASE ACTIVITY OF THE 480-KILODALTON CORRINOID PROTEINFROM METHANOSARCINA-BARKERI

Activity staining of extracts of Methanosarcina barkeri electrophorese d in polyacrylamide gels revealed an additional methylcobalamin:coenzy me M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransfer ase is the 480-kDa corrinoid protein previously identified by its meth ylation following inhibition of methyl-CoM reductase in otherwise meth anogenic cell extracts, The methylcobalamin:CoM methyltransferase acti vity of the purified 480-kDa protein increased from 0.4 to 3.8 mu mol/ min/mg after incubation with sodium dodecyl sulfate (SDS). Following S DS-polyacrylamide gel electrophoresis analysis of unheated protein sam ples, a polypeptide with an apparent molecular mass of 48 kDa which po ssessed methylcobalamin:Cor methyltransferase activity was detected, T his polypeptide migrated with an apparent mass of 41 kDa when the 480- kDa protein was heated before electrophoresis, indicating that the alp ha subunit is responsible for the activity, The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated corrinoid bound to the beta subunit of the 480-k Da protein could be demethylated by CoM, but not by homocysteine or di thiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid coul d be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 mu mol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated b y high-pressure liquid chromatography with indirect photometric detect ion, Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poore r substrates than CoM, while several others tested (including 3-mercap topropanesulfonate) did not serve as methyl accepters, These data indi cate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cof actor binding subunit during isolation.