title: The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.
abstract: A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Wenlong
authors: W
authors: Cai
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Anwesha
authors: A
authors: Goswami
authors: the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 1000050, China.
authors: Zhaoyong
authors: Z
authors: Yang
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Xiaodong
authors: X
authors: Liu
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Keith D
authors: KD
authors: Green
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Sandra
authors: S
authors: Barnard-Britson
authors: the New Modality Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., Tokyo 103-8426, Japan.
authors: Satoshi
authors: S
authors: Baba
authors: the Drug Discovery and Biomedical Technology Unit, Daiichi Sankyo RD Novare Co., Ltd., Tokyo, Japan.
authors: Masanori
authors: M
authors: Funabashi
authors: the Biologics Technology Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., Tokyo 103-8426, Japan.
authors: Koichi
authors: K
authors: Nonaka
authors: the Division of Cardiovascular Medicine and Gill Heart Institute, College of Medicine, University of Kentucky, Lexington, Kentucky 40506, and.
authors: Manjula
authors: M
authors: Sunkara
authors: the Division of Cardiovascular Medicine and Gill Heart Institute, College of Medicine, University of Kentucky, Lexington, Kentucky 40506, and.
authors: Andrew J
authors: AJ
authors: Morris
authors: the Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, 66123 Saarbrücken, Germany.
authors: Anatol P
authors: AP
authors: Spork
authors: the Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, 66123 Saarbrücken, Germany.
authors: Christian
authors: C
authors: Ducho
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Sylvie
authors: S
authors: Garneau-Tsodikova
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506.
authors: Jon S
authors: JS
authors: Thorson
authors: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506, svanlanen@uky.edu.
authors: Steven G
authors: SG
authors: Van Lanen
mesh_terms: D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001483:Base Sequence; D015195:Drug Design; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013302:Streptomyces / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D014153:Transaldolase / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry*; D014542:Uridine Monophosphate / Q000737:chemistry
chemical_list: C527229:A-102395; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; C049983:capuramycin; D013912:Threonine; D006418:Heme; D014542:Uridine Monophosphate; D014153:Transaldolase; D014529:Uridine
keywords: antibiotic resistance; antibiotics; biosynthesis; dioxygenase; natural product biosynthesis