SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone

Autor: Stephen J. Hearnshaw, Mark J. Buttner, Barrie Wilkinson, Martin Schäfer, Tung B. K. Le, Anthony Maxwell, Gregory L. Challis
Rok vydání: 2015
Předmět:
MIC
minimum inhibitory concentration
Stereochemistry
PAC
phage artificial chromosome
Streptomyces coelicolor
DNA gyrase
Article
antibiotics
Coumarins
Structural Biology
Polyketide synthase
HMBC
heteronuclear multiple bond correlation
Gene cluster
Glycosides
Enzyme Inhibitors
Molecular Biology
Biotransformation
chemistry.chemical_classification
ketoreductase
BBSRC
Biotechnology and Biological Sciences Research Council
biology
Streptomyces antibioticus
LC-MS
liquid chromatography mass spectrometry
NAD
biology.organism_classification
SD8
simocyclinone D8
Recombinant Proteins
short-chain dehydrogenase/reductase (SDR) superfamily
Anti-Bacterial Agents
Biosynthetic Pathways
3. Good health
Alcohol Oxidoreductases
Enzyme
simocyclinones
chemistry
Biochemistry
Multigene Family
Dehydratase
biology.protein
NAD+ kinase
SDR
short-chain dehydrogenase/reductase
Oxidation-Reduction
Linker
Gene Deletion
Zdroj: Journal of Molecular Biology
ISSN: 0022-2836
DOI: 10.1016/j.jmb.2015.03.019
Popis: 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.
Graphical abstract
Highlights • The ~ 75-kb simocyclinone biosynthetic cluster was expressed in a heterologous system. • SimC7 is a novel NAD(P)H-dependent ketoreductase. • SimC7 function is essential for the antibiotic activity of the DNA gyrase inhibitor simocyclinone.
Databáze: OpenAIRE
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