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Permanent link (DOI): https://doi.org/10.7939/R3RB6WC7G

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Biochemical investigations of two polyketide synthase subclasses – highly reducing/non-reducing pairs, and polyketide synthase non-ribosomal peptide synthetases. Open Access

Descriptions

Other title
Subject/Keyword
Heterologous Expression
Cytochalasin
Biosynthesis
Lovastatin
Polyketide Synthase
Cladosporin
Chaetoglobosin
Dehydrocurvularin
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Cochrane, Rachel V K
Supervisor and department
Vederas, John (Chemistry)
Examining committee member and department
Li, Liang (Chemistry)
Campbell, Robert (Chemistry)
Gerwick, William (Skaggs School of Pharmacy and Pharmaceutical Sciences)
Cairo, Christopher (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2015-12-11T14:51:41Z
Graduation date
2016-06
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Cladosporin is a polyketide metabolite that is biosynthesized by a highly reducing (HR)/non-reducing (NR) polyketide synthase (PKS) pair, wherein they catalyse head-totail condensation of acetyl and malonyl CoA units with subsequent β-keto modification. Cladosporin has recently been reported as a potent anti-malarial, acting as a nanomolar inhibitor of the Plasmodium falciparum lysyl tRNA synthetase. Sequencing of the genome of the producer organism, Cladosporium cladosporioides, allowed for identification of the cladosporin gene cluster. The HR and NR PKSs encoded therein, Cla2 and Cla3 respectively, were heterologously expressed. A series of substrate analogues were then synthesized and used in enzyme assays with Cla2. These studies showed that Cla2 could produce cladosporin from the advanced pentaketide intermediate. Cla2 also accepts analogues that contain the “unnatural” configuration at the key β- hydroxy group in the molecule, but does not recognize intermediates in which this β- hydroxy group is absent. Furthermore, Cla2 accepts analogues with shorter carbon chains, but not longer than is naturally present, suggesting a hydrophobic pocket of restricted size in the enzyme. The polyketide 10,11-dehydrocurvularin (DHC) is also biosynthesized by a HR/NR PKS pair. The genome of the producer organism, Alternaria cinerariae, was sequenced, and the DHC PKSs expressed heterologously in yeast. Availability of similar DHC-producing PKSs from Aspergillus terreus allowed us to compare biosynthetic machinery for the same metabolite in different organisms. Bioinformatic analyses suggest that sequence differences between the two sets of proteins are concentrated in specific domains, where they can be found mainly on the outer surface and specific faces of the protein. Lovastatin belongs to a group of therapeutics known as the statins, which are widely prescribed in patients with elevated levels of cholesterol. Lovastatin production is mediated by a HR PKS, LovB, in conjunction with an enoyl reductase partner, LovC, in Aspergillus terreus. During the biosynthesis of lovastatin, an interesting enzyme catalysed Diels-Alder reaction is proposed to occur in order to generate the correct stereochemistry of the decalin core. LovB contains an unusual truncated non-ribosomal peptide synthetase (NRPS) portion at its C terminus, which includes a condensation (C) domain that is thought to catalyse this reaction. In order to test this hypothesis, a series of linear hexaketide analogues, which are unable to undergo cyclization, were synthesized. These intermediates were sent to our collaborators for co-crystallization with the LovB CON domain. A 13C labeled tetraketide intermediate was also synthesized for feeding studies with LovB, to confirm its intermediacy in the biosynthesis. Chaetoglobosin A and cytochalasin E belong to a group of polyketides collectively known as the cytochalasins. This group of molecules is made by PKS-NRPS enzymes, which synthesize the polyketide backbone using their PKS domains and then add an amino acid using the NRPS portion. The polyketide-amino acid intermediate is proposed to undergo reductive release from the enzyme, followed by cyclizations and oxidation to afford the final metabolites. The exact process by which these post-PKS modifications occur is still unknown. With hopes of studying these mechanisms in more detail, synthesis of an advanced polyketide intermediate was initiated. Further elaboration of the synthesized compounds will afford the mature polyketide chain which will be sent to our collaborators in UCLA for enzymatic assays with the respective PKS-NRPS.
Language
English
DOI
doi:10.7939/R3RB6WC7G
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
Rachel V. K. Cochrane, Randy Sanichar, Gareth R. Lambkin, Béla Reiz, Wei Xu, Yi Tang, John C. Vederas. Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent. Angew. Chem. Int. Ed. Engl, 2015, DOI: 10.1002/ange.201509345.Rachel V. K. Cochrane, Zhizeng Gao, Gareth R. Lambkin, Wei Xu, Jaclyn M. Winter, Sandra L. Marcus, Yi Tang, John C. Vederas. Comparison of 10,11-dehydrocurvularin polyketide synthases from Alternaria cinerariae and Aspergillus terreus highlights key structural motifs. ChemBioChem, 2015, 17, 2479-2483.

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