title: SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.
abstract: Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone.
authors: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors: Martin
authors: M
authors: Schäfer
authors: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors: Tung B K
authors: TB
authors: Le
authors: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors: Stephen J
authors: SJ
authors: Hearnshaw
authors: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors: Anthony
authors: A
authors: Maxwell
authors: Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom.
authors: Gregory L
authors: GL
authors: Challis
authors: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors: Barrie
authors: B
authors: Wilkinson
authors: Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom. Electronic address: mark.buttner@jic.ac.uk.
authors: Mark J
authors: MJ
authors: Buttner
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D053898:Biosynthetic Pathways / Q000235:genetics; D001711:Biotransformation; D003374:Coumarins / Q000494:pharmacology; D027081:DNA Gyrase / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D006027:Glycosides / Q000494:pharmacology; D005810:Multigene Family; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D003374:Coumarins; D004791:Enzyme Inhibitors; D006027:Glycosides; D011994:Recombinant Proteins; C416814:simocyclinone D8; D009243:NAD; D000429:Alcohol Oxidoreductases; D027081:DNA Gyrase
keywords: DNA gyrase; antibiotics; ketoreductase; short-chain dehydrogenase/reductase (SDR) superfamily; simocyclinones