Polyketide natural products are secondary metabolites produced in fungi, plants and bacteria. Since their discovery, these versatile small molecules have served as pharmaceuticals in many fields of medicine. From use as antibiotics to anti-cancer agents to immunosuppresants, polyketides remain staple components in the pharmacopeia. Nature biosynthesizes members of this natural product class through use of a complex network of enzymes known as polyketide synthases. There is an interest in studying enzymatic pathways that install chemical functional groups and unique three-dimensional form in hopes of rationally modifying them to create new drug molecules. This is an attractive prospect as enzyme catalysis can be predicted by genetic examination of the pathway. In theory, swapping, deleting or inserting a catalytic domain in the pathway would offer a means of controlled alteration of the natural product. While initial efforts have led to limited success, recently the focus has been shifted to understanding the mechanistic and structural details of these pathways with the aim of improving rational pathway diversification. Three aspects of polyketide synthesis: cryptic domains, distal stereochemistry and non- canonical domain architecture; remain relatively unexplored in the polyketide literature. Cryptic domains involve the configuration of polyketide intermediates obscured by later domain action. Stereochemistry distal to the site of manipulation on the polyketide may be a factor in pathway alteration, as the new catalytic site may not accept subtle changes made by prior enzymes. Modern genetics has revealed many pathways don’t follow the rules pertaining to the presence and order of catalytic domains. Bizarre exceptions to canonical domain architecture are difficult to reproduce and predict in modified pathways. Additionally, discovering how the product is ultimately produced may offer insight and new strategies for the coupling of polyketide synthase modules to create new products. We hypothesized that all three aspects of polyketide synthases (cryptic domains, distal stereochemistry and non-canonical domain architecture) could be studied through interrogation of individual catalytic domains with synthetic, diffusible substrates. In our studies we revealed several key elements of polyketide synthases that were previously unknown in the literature. We biochemically verified that cryptic stereochemistry and geometry can be accurately predicted based on genetic patterns in a polyketide synthase. A novel, mass spectrometry-based method of quantifying enzyme turnover in polyketide synthases was developed. This new technique allowed for the direct comparison of substrates with changes in distal stereochemistry in a dehydratase domain. Single inversions in configuration in substrates were found to result in a 14- to 45-fold loss in enzyme activity. Additionally, we were able to elucidate a unique mechanism for vinylogous dehydration in the curacin A pathway. This discovery explains why the polyketide synthase is missing domains and provides a clear exception to the notion of enzyme-product co-linearity. The combined work suggests that many potential pitfalls in the rational design of polyketide synthases can be anticipated and avoided through increased knowledge of pathway mechanisms and limitations.
University of Minnesota Ph.D. dissertation. March 2016. Major: Medicinal Chemistry. Advisors: Robert Fecik, Courtney Aldrich. 1 computer file (PDF); x, 204 pages.
Biochemical Interrogation of Polyketide Ketoreductase and Dehydratase Domain Stereoselectivity, Stereospecificity and Mechanism via Synthetic, Truncated Substrates.
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