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


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.

SUBMITTER: Kim HJ 

PROVIDER: S-EPMC5831329 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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

Kim Hak Joong HJ   Liu Yung-Nan YN   McCarty Reid M RM   Liu Hung-Wen HW  

Journal of the American Chemical Society 20171107 45


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 X<sub>2</sub> (GenX<sub>2</sub>) to produce G418 during the biosynthesis of gentamicins. Reported herein, several alternative substrates and fluorinated substrate analogs were prepared to investigate the mechanism of  ...[more]

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