Structural and Biochemical Studies of Polyketide Synthase and Fatty Acid Synthase Dehydratase
Dodge, Gregory
2018
Abstract
Polyketides are chemically diverse bioactive natural products that often harbor desirable therapeutic characteristics. As such, the biological machinery used to synthesize these compounds are valuable targets for metabolic and protein engineering studies. A key characteristic of polyketide synthase (PKS) catalysis is the selective installation of many stereo- and regiocenters. To reach the full potential of PKS as a source of novel therapeutics and commodity chemicals, a molecular understanding of selective filters of each catalytic domain must be established. This thesis investigates the stereospecificity and regioselectivity of dehydratase (DH) domains from the pikromycin, curacin A, tylosin and gephyronic acid PKS pathways using a combination of biochemical assays and x-ray crystallography. These experiments establish new roles for PKS DH domains as stereoselective filters for substrate chiral centers distal from the site of catalysis, uncover previously unobserved dehydration consistent with vinylogous elimination, and establish the molecular basis for secondary isomerization. Results from these experiments facilitated the design of a novel DH/isomerase from an isomerase-inactive DH from the curacin A pathway, representing the first successful gain-of-function engineering of a PKS DH domain. Fatty acid biosynthesis is a cornerstone of metabolism in cells. Fatty acids are involved in energy storage and production, membrane construction, temperature response, and biosynthesis of many secondary metabolites. An enigmatic facet of fatty acid biosynthesis in E. coli is the presence of two functionally distinct DHs, FabA and FabZ. FabA catalyzes a secondary isomerization reaction that is the source of all unsaturated fatty acids in E. coli, while FabZ harbors only dehydratase function. This thesis establishes the molecular basis for the puzzling functional discrepancies between FabA and FabZ using functional crosslinking technology, x-ray crystallography, and molecular dynamics (MD) simulations. A high-resolution crystal structure of FabZ in complex with six AcpP subunits loaded with a C6 substrate was determined. This model facilitated MD simulations that demonstrated differential substrate preferences in agreement with published biochemical data, as well as differences in preferred substrate binding geometry such that FabZ catalyzes dehydration only whereas FabA can catalyze dehydration and subsequent isomerization. The data presented in this thesis further our understanding of PKS and will aid in future experiments to capitalize on the promise of PKS as a biological machine for the synthesis of designer polyketides and commodity chemicals. The results the from E. coli fatty acid synthase experiments answer a decades old mechanistic question and may facilitate efforts to develop antibiotics specific to bacterial fatty acid synthase.Subjects
polyketide synthase dehydratase fatty acid synthase structural biology
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