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Elucidating molecular mechanisms of actinobacterial polyketide alkaloid biosynthesis
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Ronan, Jade L. (2017) Elucidating molecular mechanisms of actinobacterial polyketide alkaloid biosynthesis. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3140724~S15
Abstract
Coelimycin P1 is a yellow-pigmented alkaloid with a unique 1,5-oxathiocane ring, produced by Streptomyces coelicolor M145. Recently, it was demonstrated that a type I modular polyketide synthase (PKS) with a C-terminal thioester reductase (TR) domain is involved in its assembly. Due to its unusual biosynthetic origin, unprecedented structure and the antibiotic activity likely associated with coelimycin A, its biosynthesis is of great interest.
To investigate the roles of the putative post-PKS tailoring enzymes (CpkG, CpkH, CpkD, ScF, CpkE and CpkI), the recombinant proteins were overproduced in E. coli, purified and incubated with various commercial and synthetic substrates. CpkG was characterised as an (S)-selective ω-transaminase with a broad substrate tolerance, responsible for the incorporation of nitrogen into the six-membered ring of coelimycin P1. Crystal structures of CpkG revealed a rare tri-domain architecture, key active site residues and provided insight into the transamination mechanism. CpkH, CpkD and ScF were all subsequently characterised as flavoproteins. Specifically, CpkH was demonstrated to catalyse an (R)-specific FAD-dependent dehydrogenation, while CpkD was shown to catalyse two FMN-dependent epoxidations. The roles of ScF, CpkE and CpkI in coelimycin biosynthesis remain to be elucidated.
Bioinformatics searches identified 22 additional actinobacterial gene clusters, which also encode modular PKSs with a TR domain and a homologue of CpkG. These have been predicted to direct the biosynthesis of both known and novel polyketide alkaloids, suggesting that reductive chain release and transamination constitute a conserved mechanism for the biosynthesis of such metabolites.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Library of Congress Subject Headings (LCSH): | Polyketides -- Synthesis, Actinobacteria, Streptomyces -- Genetics, Streptomyces coelicolor, Biosynthesis | ||||
Official Date: | July 2017 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Challis, Gregory L. | ||||
Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) ; University of Warwick. Chancellor International Scholarship | ||||
Format of File: | |||||
Extent: | xxviii, 180 leaves : illustrations, charts | ||||
Language: | eng |
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