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Enzymology of nitrogen incorporation in actinobacterial polyketide alkaloid biosynthesis
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Awodi, Ufedo Ruby (2021) Enzymology of nitrogen incorporation in actinobacterial polyketide alkaloid biosynthesis. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3756243~S15
Abstract
Numerous alkaloids of polyketide origin are known to be produced by Actinobacteria. These polyketide-alkaloids exhibit unusual structural diversity with several examples possessing mono-, di-, tri-and tetracyclic rings. Coelimycin P1 is a polyketide alkaloid which is the product of the cryptic cpk polyketide biosynthetic gene cluster in S. coelicolor M145. Its structure consists of a unique functionalized 1,5-oxathiocane attached to a piperideine ring with an extended conjugated π system which is responsible for its yellow pigmented colour. Previous bioinformatics analysis showed that the type I modular PKS encoded by the cpk gene cluster contains a C-terminal thioester reductase (TR) domain proposed to catalyse the reductive release of the fully assembled polyketide chain, converting it to the corresponding aldehyde. A gene encoding a putative pyridoxal phosphate (PLP)-dependent transaminase (CpkG) was also identified within the cpk gene cluster. CpkG was predicted to convert the aldehyde product of the type I PKS to the corresponding amine prior to subsequent transformations leading to the assembly of coelimycin P1.
The TR domain and CpkG were overproduced as recombinant proteins in E. coli and purified to investigate their enzymatic activity. The TR domain was shown to catalyse the NADH-dependent reduction of structural analogues of the fully assembled polyketide chain (commercially available short to medium chain acyl-CoAs). It preferentially utilized NADH over NADPH. Recombinant CpkG was shown to catalyse the PLP-dependent transamination of short to medium chain aldehydes to their corresponding amines, utilizing both L-and D-alanine as well as L-glutamate.
Results obtained herein, show that the coelimycin P1 biosynthetic pathway (involving reduction of the thioester bond to the aldehyde followed by reductive amination to yield the amine) is distinct from previously described nitrogen incorporation pathways in alkaloid biosynthesis which typically involve either L-lysine or L-ornithine as precursors; with the entire carbon backbone of the nitrogen heterocycle being derived from the amino acid.
Bioinformatics analysis carried out during subsequent work, on sequenced actinobacterial genomes deposited in the GenBank database, querying for biosynthetic gene clusters harbouring genes encoding for a type I modular PKS with a TR domain as well as a CpkG homologue, identified 22 clusters likely to be responsible for the assembly of polyketide-alkaloids. This suggests that NAD(P)-dependent reductive chain release and PLP-dependent transamination is a conserved feature of polyketide-alkaloid biosynthesis.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Library of Congress Subject Headings (LCSH): | Actinobacteria, Polyketides, Alkaloids -- Synthesis | ||||
Official Date: | October 2021 | ||||
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: | University of Warwick. Chancellor's International Scholarship | ||||
Extent: | xxi, 138 leaves : illustrations, charts | ||||
Language: | eng |
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