Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.699587
Title: Functional and structural analysis of the angucyclinone ketoreductase SimC7
Author: Schäfer, Martin
ISNI:       0000 0004 5990 3642
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2016
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Abstract:
The natural product simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The biosynthetic simocyclinone (sim) 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. SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins, was proposed to supply the dehydratase activity missing from two modules of the polyketide synthase. In this study, I report the structure and function of recombinant S. antibioticus SimC7. Because the natural simocyclinone producer is genetically intractable, the ~72-kb sim cluster was isolated on a single phage artificial chromosome (PAC) clone for heterologous production in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in production of a novel simocyclinone, 7-oxo-SD8, which carried a normal tetraene linker but was altered in the angucyclinone. I demonstrate that SimC7 is an NAD(P)H-dependent polyketide ketoreductase that catalyses the reduction of the C-7 carbonyl of the angucyclinone and the resulting hydroxyl group is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct catalytic mechanism. To investigate this possibility, I determined the structures of SimC7 alone, in complex with NADP+, and in complex with NADP+ and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad (Ser-Tyr-Lys), including the active site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active site mutants, my data suggest SimC7 catalyses a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.699587  DOI: Not available
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