Combinatorial biosynthesis of polyketides through engineering the respective biosynthetic pathways represents a promising approach to natural products discovery. However, a lack of information on substrate and stereochemical specificity of polyketide synthases (PKS) currently hinders these efforts. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase (KR) and dehydratase (DH) domains of PKS. PKS KRs stereoselectively reduce the β-keto chain intermediate while dictating the orientation of the α-substituent. The DHs of PKS, which generate an α,β-unsaturated bond through dehydration of a β-alcohol, have extremely high stereospecficity towards substrates and can prematurely terminate the nascent polyketide when presented with unnatural substrates. We have developed a chemical tool-based approach which was verified using KR and DH of pikromycin PKS module 2 (PikKR2) as a model system. Triketide substrate mimics were designed to increase stability (incorporating a non-hydrolyzable thioether linkage) and minimize non-essential functionality (truncating the phosphopantetheinyl arm). The identities of reduction and dehydration products as well as steady-state kinetic parameters were revealed by a LC-MS/MS analysis of synthetic standards. Additionally, the substrate specificity was interrogated with a systematic series of synthetic triketides containing altered stereogenic centers. Furthermore, the mechanism of PikDH2 catalyzed dehydration was investigated by site-directed mutagenesis, evaluation of the pH dependence of catalytic efficiency (Vmax/KM), and through kinetic characterization of a mechanism-based inhibitor.
University of Minnesota Ph.D. dissertation.July 2015. Major: Medicinal Chemistry. Advisors: Robert Fecik, Courtney Aldrich. 1 computer file (PDF); xi, 316 pages.
Deciphering Cryptic Stereochemistry in Polyketide Biosynthesis and Functional Characterization of β-processing Domains in Pikromycin Synthase.
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