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

Overview

Journal J Am Chem Soc

Publisher American Chemical Society

Specialty Chemistry

Date 2017 Nov 2

PMID 29091410

Citations 22

Authors

Hak Joong Kim

Yung-Nan Liu

Reid M McCarty

Hung-Wen Liu

Affiliations

Soon will be listed here.

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 X (GenX) 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 GenX 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.

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References

Blaszczyk A, Silakov A, Zhang B, Maiocco S, Lanz N, Kelly W . Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase. J Am Chem Soc. 2016; 138(10):3416-26. DOI: 10.1021/jacs.5b12592. View

Sato S, Kudo F, Kim S, Kuzuyama T, Eguchi T . Methylcobalamin-Dependent Radical SAM C-Methyltransferase Fom3 Recognizes Cytidylyl-2-hydroxyethylphosphonate and Catalyzes the Nonstereoselective C-Methylation in Fosfomycin Biosynthesis. Biochemistry. 2017; 56(28):3519-3522. DOI: 10.1021/acs.biochem.7b00472. View

Parent A, Guillot A, Benjdia A, Chartier G, Leprince J, Berteau O . The B-Radical SAM Enzyme PoyC Catalyzes Valine C-Methylation during Polytheonamide Biosynthesis. J Am Chem Soc. 2016; 138(48):15515-15518. PMC: 5410653. DOI: 10.1021/jacs.6b06697. View

Kim H, McCarty R, Ogasawara Y, Liu Y, Mansoorabadi S, LeVieux J . GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme. J Am Chem Soc. 2013; 135(22):8093-6. PMC: 3796153. DOI: 10.1021/ja312641f. View

Ruszczycky M, Liu H . Mechanistic Enzymology of the Radical SAM Enzyme DesII. Isr J Chem. 2016; 55(3-4):315-324. PMC: 5021314. DOI: 10.1002/ijch.201400130. View

Kim J, Darley D, Buckel W, Pierik A . An allylic ketyl radical intermediate in clostridial amino-acid fermentation. Nature. 2008; 452(7184):239-42. DOI: 10.1038/nature06637. View

Bauerle M, Schwalm E, Booker S . Mechanistic diversity of radical S-adenosylmethionine (SAM)-dependent methylation. J Biol Chem. 2014; 290(7):3995-4002. PMC: 4326810. DOI: 10.1074/jbc.R114.607044. View

Huang C, Huang F, Moison E, Guo J, Jian X, Duan X . Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex. Chem Biol. 2015; 22(2):251-61. PMC: 4340712. DOI: 10.1016/j.chembiol.2014.12.012. View

Hu K, Werner W, Allen K, Wang S . Investigation of enzymatic C-P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy. Magn Reson Chem. 2015; 53(4):267-72. PMC: 4656027. DOI: 10.1002/mrc.4190. View

10.

Buckel W . Radical and electron recycling in catalysis. Angew Chem Int Ed Engl. 2009; 48(37):6779-87. DOI: 10.1002/anie.200900771. View

11.

Allen K, Wang S . Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217. Biochim Biophys Acta. 2014; 1844(12):2135-44. PMC: 4362910. DOI: 10.1016/j.bbapap.2014.09.009. View

12.

Allen K, Wang S . Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis. Arch Biochem Biophys. 2013; 543:67-73. PMC: 4048823. DOI: 10.1016/j.abb.2013.12.004. View

13.

Zhang Q, van der Donk W, Liu W . Radical-mediated enzymatic methylation: a tale of two SAMS. Acc Chem Res. 2011; 45(4):555-64. PMC: 3328197. DOI: 10.1021/ar200202c. View

14.

Benjdia A, Pierre S, Gherasim C, Guillot A, Carmona M, Amara P . The thiostrepton A tryptophan methyltransferase TsrM catalyses a cob(II)alamin-dependent methyl transfer reaction. Nat Commun. 2015; 6:8377. PMC: 4632189. DOI: 10.1038/ncomms9377. View

15.

Frey P, Hegeman A, Ruzicka F . The Radical SAM Superfamily. Crit Rev Biochem Mol Biol. 2008; 43(1):63-88. DOI: 10.1080/10409230701829169. View

16.

Bridwell-Rabb J, Zhong A, Sun H, Drennan C, Liu H . A B-dependent radical SAM enzyme involved in oxetanocin A biosynthesis. Nature. 2017; 544(7650):322-326. PMC: 5398914. DOI: 10.1038/nature21689. View

17.

Lin G, Choi S, Ruszczycky M, Liu H . Mechanistic Investigation of the Radical S-Adenosyl-L-methionine Enzyme DesII Using Fluorinated Analogues. J Am Chem Soc. 2015; 137(15):4964-7. PMC: 4862307. DOI: 10.1021/jacs.5b02545. View

18.

Ostash B, Doud E, Lin C, Ostash I, Perlstein D, Fuse S . Complete characterization of the seventeen step moenomycin biosynthetic pathway. Biochemistry. 2009; 48(37):8830-41. PMC: 2747051. DOI: 10.1021/bi901018q. View

19.

Pierre S, Guillot A, Benjdia A, Sandstrom C, Langella P, Berteau O . Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes. Nat Chem Biol. 2012; 8(12):957-9. DOI: 10.1038/nchembio.1091. View

20.

Grillo M, Colombatto S . S-adenosylmethionine and its products. Amino Acids. 2007; 34(2):187-93. DOI: 10.1007/s00726-007-0500-9. View