Design and synthesis of polyketide-based labels for polyketide synthase thioesterase and ketoreductase domains
2010-10
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Design and synthesis of polyketide-based labels for polyketide synthase thioesterase and ketoreductase domains
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2010-10
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Abstract
Polyketides are a diverse class of natural products with a wide range of biological and pharmacological activities. Polyketides are biosynthesized by modular multienzyme complexes, polyketide synthases (PKSs), through sequential condensation of simple carboxylic acid building blocks. Due to multi-drug resistant bacteria becoming a growing public health problem, there is increased interest to exploit these systems to produce novel molecules and drug leads through combinatorial biosynthesis; however, determining substrate selectivity or stereospecificity is crucial for understanding PKS catalytic domains. Of recent interest have been the thiosesterase (TE) enzyme domain, which is responsible for transesterification reactions and cyclization, and the ketoreductase (KR) enzyme domain, which controls the stereochemistry in the reduction pathway where the รข-ketone moiety of a polyketide is converted into an alcohol.
TE and KR crystal structures have been solved and now interest in harnessing their chemical potential is being explored. Several groups have embarked upon the challenge of engineering PKS for combinatorial biosynthesis. Their efforts have ranged from genetic engineering and heterologous expression to understanding the structure and function of modular PKSs; however, a lack of structure-based understanding for substrate specificity for nearly all of the catalytic domains creates several challenges for the use of PKSs in combinatorial biosynthesis. The objective of this research is to design polyketide-based labels in order to understand this relationship. Initial studies carried out by our lab provided structural and mechanistic insights towards engineering Pik TE for combinatorial biosynthesis; however, more information is needed, due to Pik TE structure-based dependence, to determine whether macrolactonization or hydrolytic activity will be observed. Therefore, here we present the successful design and synthesis of polyketide-based labels for studies with the TE and KR domains.
The goals of this research can be divided in two parts: chloromethylketone affinity labels were design and synthesized for PKS TE domains, and CoA-analogs were synthesized through modification of CoA with chloromethylketones and vinylketones for PKS ACP-containing didomains, specifically KR-ACP and ACP-TE didomains. We observed the successful labeling of KR-ACP and ACP-TE didomains and co-crystallization trials are still on-going.
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University of Minnesota Ph.D. dissertation. October, 2010. Major: Chemistry. Advisors: Dr. Mark D. Distefano, Dr. Robert A. Fecik. 1 computer file (PDF); xi, 134 pages, appendices p. 98-134.
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Leggans, Erick K.L.S.. (2010). Design and synthesis of polyketide-based labels for polyketide synthase thioesterase and ketoreductase domains. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/100272.
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