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Investigation of Rieske non-heme iron-dependent oxygenase-catalyzed oxidative carbocyclization reactions in prodiginine alkaloid biosynthesis
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Perry, Christopher (2019) Investigation of Rieske non-heme iron-dependent oxygenase-catalyzed oxidative carbocyclization reactions in prodiginine alkaloid biosynthesis. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3491685~S15
Abstract
Streptorubin B and metacycloprodigiosin are specialized metabolites belonging to the prodiginine family produced by S. coelicolor A3(2) and S. longispororuber respectively. The final step in their biosynthesis is an oxidative carbocyclization reaction of undecylprodigiosin (scheme 1). RedG is a Rieske non-heme iron dependent oxygenase that catalyzes the oxidative carbocyclization reaction at C-7’ of the undecylprodigiosin alkyl chain to furnish streptorubin B. McpG is 75 % similar in sequence to RedG and catalyzes an analogous cyclization at C-9' of undecylprodigiosin to form metacycloprodigiosin.
Scheme 1: The Rieske oxygenase catalyzed oxidative carbocyclization reactions of undecylprodigiosin to form streptorubin B and metacycloprodigiosin.
The mechanism and stereochemical course of the McpG catalyzed cyclization reaction has been investigated via mutasynthesis. The entire metacycloprodigiosin biosynthetic gene cluster was captured from the genome of S. longispororuber using yeast-mediated transform associated recombination (TAR) cloning. The mcp cluster was heterologously expressed in S. albus and production of metacycloprodigiosin was confirmed. TAR was then used to create an in-frame deletion of a PKS-reductase fusion gene involved in the biosynthesis of 2- undecylpyrrole (2-UP), resulting in loss of metacycloprodigiosin production, which could be restored by feeding synthetic 2-UP. A range of 2-UP analogues were synthesized bearing various functional groups designed to probe different aspects of the McpG mechanism by feeding to the deletion mutant expressing an extra copy of mcpH and mcpG. The results showed that McpG catalyzes a direct cyclization reaction and does not catalyze the formation of a hydroxylated undecylprodigiosin intermediate via rebound onto the iron-oxo species. McpG was able to cyclize different alkyl chain lengths, but did not accept substrates with additional branching or adjacent π-bonds, and catalyzes cyclization with inversion of stereochemistry at C-9’ of the alkyl chain. RedG was over-expressed, purified and the Rieske [2Fe-2S] cluster characterized by UV-Vis spectroscopy and chemically reconstituted in vitro.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Library of Congress Subject Headings (LCSH): | Alkaloids -- Synthesis, Metabolites, Biosynthesis, Ring formation (Chemistry), Oxygenases, Catalysis, Streptomyces | ||||
Official Date: | September 2019 | ||||
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: | Engineering and Physical Sciences Research Council | ||||
Format of File: | |||||
Extent: | xxvi, 217 leaves : illustrations, charts | ||||
Language: | eng |
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