Browsing by Subject "Polyketide Synthase"

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    Deciphering Cryptic Stereochemistry in Polyketide Biosynthesis and Functional Characterization of β-processing Domains in Pikromycin Synthase
    (2015-07) Li, Yang
    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.
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    Design and synthesis of polyketide-based labels for polyketide synthase thioesterase and ketoreductase domains
    (2010-10) Leggans, Erick K.L.S.
    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 â-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.