title: Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri.
abstract: Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa corrinoid protein previously identified by its methylation following inhibition of methyl-CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha 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-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high-pressure liquid chromatography with indirect photometric detection. Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cofactor binding subunit during isolation.
authors.affiliation: Department of Microbiology, Ohio State University, Columbus 43210, USA.
authors.forename: T C
authors.initials: TC
authors.lastname: Tallant
authors.forename: J A
authors.initials: JA
authors.lastname: Krzycki
mesh_terms: D000085:Acetates / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D045728:Corrinoids; D006847:Hydrocarbons, Iodinated / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000254:growth & development; D008744:Methylamines / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011166:Porphyrins; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D000085:Acetates; D019843:Archaeal Proteins; D045728:Corrinoids; D006847:Hydrocarbons, Iodinated; D007527:Isoenzymes; D008744:Methylamines; D011166:Porphyrins; D013438:Sulfhydryl Compounds; C014055:methyl iodide; D008780:Methyltransferases; C023894:methylcobalamin-coenzyme M methyltransferase; C023336:trimethylamine