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Permanent link (DOI): https://doi.org/10.7939/R3SQ8QR76

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Investigation of the biosynthesis of lovastatin and cytochalasin E, two fungal polyketides assembled by iterative PKS-NRPS enzymes Open Access

Descriptions

Other title
Subject/Keyword
polyketide
PKS-NRPS
cytochalasin E
lovastatin
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Thuss, Justin A. J.
Supervisor and department
Dr. John C. Vederas
Examining committee member and department
Dr. Russell Kerr (external)
Dr. Fredrick West (Chemistry)
Dr. Robert Campbell (Chemistry)
Dr. Liang Li (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2015-09-25T10:32:13Z
Graduation date
2015-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Cytochalasin E and lovastatin are two fungal polyketides that have seen extensive use as an angiogenesis inhibitor and as a cholesterol-lowering agent, respectively. These structurally divergent compounds are synthesized by polymerization of acetate units with concomitant functionalization by two different polyketide synthases (PKSs), CcsA and LovB. Both of these fungal synthases belong to a unique class of enzymes called fungal iterative polyketide synthase-nonribosomal peptide synthetase hybrids (fungal iterative PKS-NRPSs). These enzymes are unique, as the PKS component contains a suite of reactive domains that are similar in architecture to fatty acid synthases (FASs) but construct much more functionalized acyl chains than the long saturated lipids made by FASs. The NRPS region typically facilitates attachment of an amino acid to the acyl chain and release from the synthase. The diversity of the natural products produced from PKS-NRPSs is astonishing, yet very little is understood about how the domains operate. The iterative nature of the domains means that they must display “inherent selectivity” that results in partial reduction on some sections of the polyketide chain. This is sometimes referred to as the “programming” of the synthase. This thesis explores the biosynthesis of cytochalasin E by first investigating the biogenesis of its unique macrocyclic carbonate moiety. Its formation is catalyzed by CcsB, a Baeyer-Villigerase encoded in the gene cluster for cytochalasin E in Aspergillus clavatus. CcsB was heterologously expressed, purified and assayed in vitro using chemically synthesized substrates. CcsB formally catalyzes a double Baeyer-Villiger reaction and its mechanism was investigated using isotope labeling. Formation of cytochalasin E’s carbon backbone was investigated by expressing CcsA in vitro. Attempts towards the synthesis of a late-stage, fully functionalized PKS intermediate are detailed. The unusual activity and role of the reductase domain (R) of the NRPS region of CcsB was examined as well. The programming of lovastatin biosynthesis was studied by focusing on the activity of one domain of the PKS-NRPS, namely the methyltransferase domain (MT). The MT domain of LovB is active during one single round of chain elongation in the process of lovastatin biosynthesis. This activity was explored by expressing the megasynthase LovB in vitro and assaying the methylation activity using synthetic intermediates. Results indicate that the selection is at the substrate level and may be kinetically controlled.
Language
English
DOI
doi:10.7939/R3SQ8QR76
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
Hu, Y.; Dietrich, D.; Xu, W.; Patel, A.; Thuss J. A. J.; Wang, J.; Yin, W.-B.; Qiao, K.; Houk, K. N.; Vederas J. C.; Tang, Y. Nat Chem Biol 2014, 10, 552.

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