Publications

• 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 H.J., Liu Y.N., McCarty R.M., Liu H.W.

  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 ... >> More

  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. << Less

  J Am Chem Soc 139:16084-16087(2017) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Studies of GenK and OxsB, two B₁₂-dependent radical SAM enzymes involved in natural product biosynthesis.

  Lee Y.H., Liu H.W.

  The B₁₂-dependent radical SAM enzymes are an emerging subgroup of biological catalysts that bind a cobalamin cofactor in addition to the canonical [Fe₄S₄] cluster characteristic of radical SAM enzymes. Most of the B₁₂-dependent radical SAM enzymes that h ... >> More

  The B₁₂-dependent radical SAM enzymes are an emerging subgroup of biological catalysts that bind a cobalamin cofactor in addition to the canonical [Fe₄S₄] cluster characteristic of radical SAM enzymes. Most of the B₁₂-dependent radical SAM enzymes that have been characterized mediated methyltransfer reactions; however, a small number are known to catalyze more diverse reactions such as ring contractions. Thus, Genk is a methyltransferase from the gentamicin C biosynthetic pathway, whereas OxsB catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphates to generate an oxetane aldehyde during the biosynthesis of oxetanocin A. The preparation and in vitro characterization of such enzymes is complicated by the presence of two redox sensitive cofactors in addition to challenges in obtaining soluble protein for study. This chapter describes expression, purification and assay methodologies for GenK and OxsB highlighting the use of denaturation/refolding protocols for solubilizing inclusion bodies as well as the use of cluster assembly and cobalamin uptake machinery during in vivo expression. << Less

  Methods Enzymol 669:71-90(2022) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme.

  Kim H.J., McCarty R.M., Ogasawara Y., Liu Y.N., Mansoorabadi S.O., LeVieux J., Liu H.W.

  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 mechanism ... >> More

  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. << Less

  J Am Chem Soc 135:8093-8096(2013) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.