No abstract

The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-L-methionine (SAM) superfamily was previously predicted based on 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 one equivalent 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. Based on 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.

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.

UDP-D-apiose/UDP-D-xylose synthase (AXS) catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-apiose and UDP-D-xylose. An acetyl-protected phosphonate analogue of UDP-D-apiose was synthesized and used in an in situ HPLC assay to demonstrate, for the first time, the ability of AXS to interconvert the two reaction products. Density functional theory calculations provided insight into the energetics of this process and the apparent inability of AXS to catalyze the conversion of UDP-D-xylose to UDP-D-apiose. The data suggest that this observation is unlikely to be due to an unfavorable equilibrium, but rather substrate inhibition by the most stable chair conformation of UDP-D-xylose. The detection of xylose cyclic phosphonate as the turnover product uncovers significant new detail about the AXS-catalyzed reaction and supports the proposed retroaldol-aldol mechanism of catalysis.