GenK-catalyzed C-6′ methylation in the biosynthesis of gentamicin: Isolation and characterization of a cobalamin-dependent radical SAM enzyme

Hak Joong Kim, Reid M. McCarty, Yasushi Ogasawara, Yung Nan Liu, Steven O. Mansoorabadi, Jake Levieux, Hung Wen Liu

Research output: Contribution to journalArticle

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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 sp3 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.

Original languageEnglish
Pages (from-to)8093-8096
Number of pages4
JournalJournal of the American Chemical Society
Volume135
Issue number22
DOIs
Publication statusPublished - 2013 Jun 5
Externally publishedYes

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Methylation
Biosynthesis
Vitamin B 12
Gentamicins
Methionine
Enzymes
Antibiotics
Bioinformatics
Stoichiometry
Labeling
Isotopes
Methyltransferases
Aminoglycosides
Experiments
Carbon
Isotope Labeling
Homocysteine
Computational Biology
Anti-Bacterial Agents

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

GenK-catalyzed C-6′ methylation in the biosynthesis of gentamicin : Isolation and characterization of a cobalamin-dependent radical SAM enzyme. / Kim, Hak Joong; McCarty, Reid M.; Ogasawara, Yasushi; Liu, Yung Nan; Mansoorabadi, Steven O.; Levieux, Jake; Liu, Hung Wen.

In: Journal of the American Chemical Society, Vol. 135, No. 22, 05.06.2013, p. 8093-8096.

Research output: Contribution to journalArticle

Kim, Hak Joong ; McCarty, Reid M. ; Ogasawara, Yasushi ; Liu, Yung Nan ; Mansoorabadi, Steven O. ; Levieux, Jake ; Liu, Hung Wen. / GenK-catalyzed C-6′ methylation in the biosynthesis of gentamicin : Isolation and characterization of a cobalamin-dependent radical SAM enzyme. In: Journal of the American Chemical Society. 2013 ; Vol. 135, No. 22. pp. 8093-8096.
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