title: Genome mining of the biosynthetic gene cluster of the polyene macrolide antibiotic tetramycin and characterization of a P450 monooxygenase involved in the hydroxylation of the tetramycin B polyol segment.
abstract: A polyene macrolide antibiotic tetramycin biosynthetic gene cluster was identified by genome mining and isolated from Streptomyces hygrospinosus var. beijingensis. Genetic and in silico analyses gave insights into the mechanism of biosynthesis of tetramycin, and a model of the tetramycin biosynthetic pathway is proposed. Inactivation of a cytochrome P450 monooxygenase gene, tetrK, resulted in the production of a tetramycin B precursor: tetramycin A, which lacks a hydroxy group in its polyol region. TetrK was subsequently overexpressed heterologously in E. coli with a His(6)  tag, and purified TetrK efficiently hydroxylated tetramycin A to afford tetramycin B. Kinetic studies revealed no inhibition of TetrK by substrate or product. Surprisingly, sequence-alignment analysis showed that TetrK, as a hydroxylase, has much higher homology with epoxidase PimD than with hydroxylases NysL and AmphL. The 3D structure of TetrK was then constructed by homology modeling with PimD as reference. Although TetrK and PimD catalyzed different chemical reactions, homology modeling indicated that they might share the same catalytic sites, despite also possessing some different sites correlated with substrate binding and substrate specificity. These findings offer good prospects for the production of improved antifungal polyene analogues.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Cao
authors.forename: Fen
authors.initials: F
authors.lastname: Yao
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zheng
authors.forename: Dongbing
authors.initials: D
authors.lastname: Cui
authors.forename: Yucheng
authors.initials: Y
authors.lastname: Shao
authors.forename: Changxiong
authors.initials: C
authors.lastname: Zhu
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.forename: Delin
authors.initials: D
authors.lastname: You
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006900:Hydroxylation; D018942:Macrolides / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011090:Polyenes / Q000378:metabolism*; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D018942:Macrolides; D011090:Polyenes; D003577:Cytochrome P-450 Enzyme System