Methylcobamide:coenzyme M methyltransferase isozymes from Methanosarcina barkeri. Physicochemical characterization, cloning, sequence analysis, and heterologous gene expression.
A comparative study was made on the physicochemical characteristics of two isozymes of methylcobamide:- coenzyme M methyltransferase (MT2). Both isozymes catalyzed S-methylation of 2-thioethanesulfonate (coenzyme M) and exhibited similar apparent Km values for coenzyme M of 35 microM (MT2-A) and 20 microM (MT2-M). Weak binding to methylcobalamin was indicated by the apparent Km of 14 mM for both isozymes. Cob(I)alamin was established as the major product of the reaction, demonstrating heterolytic cleavage of the methylcobamide carbon-cobalt bond. The isozymes were shown to be zinc-containing metalloproteins. Metal ion chelators strongly inhibited both isozymes. A variety of coenzyme M analogs were tested for activity and/or inhibition. One alternative substrate 3-mercaptopropionate was discovered, with apparent Km 9 mM (MT2-A) and 10 mM (MT2-M). The results suggested an active site geometry in which coenzyme M is bound both by S-coordination to zinc, and electrostatic interaction of the sulfonate with a cationic group on the enzyme. Methanosarcina barkeri genes cmtA and cmtM encoding both isozymes were cloned and sequenced. Both genes encoded proteins with 339 amino acids and predicted molecular masses of 36-37 kDa. Active forms of both isozymes were expressed in Escherichia coli. A conserved segment with the potential for metal binding was found. The possibility of zinc involvement in catalysis of coenzyme M methylation is considered.
LeClerc
G M
GM
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Grahame
D A
DA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002614:Chelating Agents / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000235:genetics*; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
D002614:Chelating Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D007527:Isoenzymes; D008780:Methyltransferases; C098666:methylcobamide-coenzyme M methyltransferase; D015032:Zinc