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Investigations of Fungal Highly Reducing Polyketide Synthases: The Biosyntheses of Lovastatin and Hypothemycin Open Access


Other title
acyl carrier protein
natural products chemistry
biological Diels-Alder reaction
Type of item
Degree grantor
University of Alberta
Author or creator
Norquay, Amy K
Supervisor and department
Vederas, John (Chemistry)
Examining committee member and department
West, Frederick (Chemistry)
Garneau, Sylvie (University of Kentucky College of Pharmacy)
Derda, Ratmir (Chemistry)
Williams, Florence (Chemistry)
Department of Chemistry

Date accepted
Graduation date
2017-11:Fall 2017
Doctor of Philosophy
Degree level
Fungal highly reducing polyketide synthases (HR-PKSs) are large, multi-domain enzymes central to the biosynthesis of fungal polyketides. They assemble complex secondary metabolites, called polyketides, through the condensation of two-carbon acetate units and tailoring of the functional groups at each round of chain extension. Two such fungal polyketides are lovastatin, a cholesterol-lowering therapeutic, and hypothemycin, an anti-cancer kinase inhibitor. Studies into the biosynthesis of these two natural products were undertaken to expand our understanding of the complex processes performed by HR-PKS enzymes. Firstly, the hypothesized reaction sequence of HR-PKS enzymes for these metabolites, proposed based on their similarity to fatty acid synthases, had never been fully proven experimentally. In our study of hypothemycin biosynthesis, we synthesized the proposed enzyme bound intermediates as N-acetylcysteamine thioesters with 13C labels to use in incorporation assays with purified enzymes. We observed conversion of the partially assembled intermediates into intact PKS products, confirming their intermediacy and validating our understanding of HR-PKS reaction sequences. In order to extrapolate these findings to other systems, the proposed intermediates of lovastatin were also synthesized, and enzymatic assays are forthcoming. Secondly, it has been proposed that an enzyme-catalyzed Diels-Alder cyclization is a key step in the assembly of the lovastatin intermediate dihydromonacolin L by the HR-PKS LovB. In order to study this LovB-catalyzed cyclization, two potential substrates were synthesized and various fragments of LovB were expressed heterologously. NMR techniques were developed to determine the products of the cyclization assays. By monitoring the conversion of the hexaketide triene to the enzymatic decalin product, it was determined that the Diels-Alder activity was preserved in the LovB fragment where the condensation (CON) domain was deleted (LovBΔCON). Therefore our hypothesis that the CON domain is the Diels-Alderase is incorrect. It was also found that LovB did not catalyze the cyclization of a proposed heptaketide tetraene intermediate, and therefore the substrate scope of this conversion is still being investigated in order to better understand how this reaction occurs in nature. Finally, the acyl carrier protein domains of both LovB, and the hypothemycin polyketide synthase, Hpm8, were expressed as stand-alone proteins. Our goal was to obtain a solution structure using NMR spectroscopy but the inherent properties of the protein made that impossible. Herein I explore the properties of these domains and discuss other possible ways we can use the expressed proteins to better understand the HR-PKS class of enzymes.
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