title: Purification and characterization of dimethylamine:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barkeri Fusaro.
abstract: 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 Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 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 B12/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.
authors.affiliation: Department of Microbiology, Faculty of Science, University of Nijmegen, The Netherlands. roelw@sci.kun.nl
authors.forename: R W
authors.initials: RW
authors.lastname: Wassenaar
authors.forename: J T
authors.initials: JT
authors.lastname: Keltjens
authors.forename: C
authors.initials: C
authors.lastname: van der Drift
authors.forename: G D
authors.initials: GD
authors.lastname: Vogels
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003038:Cobamides / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007700:Kinetics; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000254:growth & development; D008744:Methylamines / Q000378:metabolism; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D008956:Models, Chemical; D008970:Molecular Weight
chemical_list: D003038:Cobamides; D008744:Methylamines; C113290:DMA-methyltransferase; D008780:Methyltransferases; C103170:dimethylamine-coenzyme M methyltransferase; C023336:trimethylamine