Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl- l -methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration

Hak Joong Kim, Yung Nan Liu, Reid M. McCarty, Hung Wen Liu

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12 Citations (Scopus)

Abstract

Many cobalamin (Cbl)-dependent radical S-adenosyl-l-methionine (SAM) methyltransferases have been identified through sequence alignment and/or genetic analysis; however, few have been studied in vitro. GenK is one such enzyme that catalyzes methylation of the 6′-carbon of gentamicin X2 (GenX2) to produce G418 during the biosynthesis of gentamicins. Reported herein, several alternative substrates and fluorinated substrate analogs were prepared to investigate the mechanism of methyl transfer from Cbl to the substrate as well as the substrate specificity of GenK. Experiments with deuterated substrates are also shown here to demonstrate that the 6′-pro-R-hydrogen atom of GenX2 is stereoselectively abstracted by the 5′-dAdo· radical and that methylation occurs with retention of configuration at C6′. Based on these observations, a model of GenK catalysis is proposed wherein free rotation of the radical-bearing carbon is prevented and the radical SAM machinery sits adjacent rather than opposite to the Me-Cbl cofactor with respect to the substrate in the enzyme active site.

Original languageEnglish
Pages (from-to)16084-16087
Number of pages4
JournalJournal of the American Chemical Society
Volume139
Issue number45
DOIs
Publication statusPublished - 2017 Nov 15
Externally publishedYes

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ASJC Scopus subject areas

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

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