title: Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex.
abstract: Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People's Republic of China.
authors: Chuan
authors: C
authors: Huang
authors: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
authors: Fanglu
authors: F
authors: Huang
authors: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
authors: Eileen
authors: E
authors: Moison
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People's Republic of China.
authors: Junhong
authors: J
authors: Guo
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People's Republic of China.
authors: Xinyun
authors: X
authors: Jian
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People's Republic of China.
authors: Xiaobo
authors: X
authors: Duan
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People's Republic of China; Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of Biotechnology, Wuhan 430075, People's Republic of China.
authors: Zixin
authors: Z
authors: Deng
authors: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK. Electronic address: pfl10@cam.ac.uk.
authors: Peter F
authors: PF
authors: Leadlay
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, People's Republic of China. Electronic address: yhsun@whu.edu.cn.
authors: Yuhui
authors: Y
authors: Sun
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D055162:Biocatalysis; D004926:Escherichia coli / Q000378:metabolism; D005839:Gentamicins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D005839:Gentamicins; D011994:Recombinant Proteins; C012331:gentamicin X(2); C030880:gentamicin A; D010088:Oxidoreductases; D008780:Methyltransferases; D000637:Transaminases
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