title: GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme.
abstract: The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-l-methionine (SAM) superfamily was previously predicted on the basis of bioinformatic analysis. A number of these are Cbl-dependent methyltransferases, but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the in vitro activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated sp(3) carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that 1 equiv each of 5'-deoxyadenosine and S-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the S-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. On the basis of these results, one mechanistic possibility for the GenK reaction can be ruled out, and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Hak Joong
authors.initials: HJ
authors.lastname: Kim
authors.forename: Reid M
authors.initials: RM
authors.lastname: McCarty
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ogasawara
authors.forename: Yung-nan
authors.initials: YN
authors.lastname: Liu
authors.forename: Steven O
authors.initials: SO
authors.lastname: Mansoorabadi
authors.forename: Jake
authors.initials: J
authors.lastname: LeVieux
authors.forename: Hung-wen
authors.initials: HW
authors.lastname: Liu
mesh_terms: D055162:Biocatalysis; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D005839:Gentamicins / Q000096:biosynthesis* / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014805:Vitamin B 12 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D005839:Gentamicins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D014805:Vitamin B 12