Chemical and Biochemical Interrogation of Molecular Specificity in ModularPolyketide Synthases.

[摘要] Polyketide natural products represent a diverse set of chemical entities that are prized by organic and medicinal chemists for their complex molecular architectures and unique pharmacological properties. These natural products are generated as secondary metabolites in a variety of marine and terrestrial microbial sources through the decarboxylative condensation of coenzyme A (CoA) esters of simple malonic acids. These condensation reactions occur on large, modular enzyme complexes called polyketide synthases (PKSs). Two prime examples of these unique multifunctional enzyme systems are the 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) PKS, which are responsible for the biosynthesis of the erythromycin and pikromycin aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing and macrocyclization. Due to the modular architecture of bacterial type I PKSs, rational bioengineering of these enzymes has opened up an avenue toward the rapid generation of polyketide chemical diversity through chemoenzymatic synthesis and combinatorial biosynthesis. Realization of this goal, however, requires a detailed understanding of molecular specificity in the catalytic domains of these PKS enzymes. Toward this goal, this dissertation describes the development of synthetic methodologies to access late-stage polyketide chain-elongation intermediates from the DEBS and Pik systems and their subsequent employment in biochemical studies with engineered PKS modules. Specifically, the native pentaketide intermediate for the DEBS system was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in the final engineered monomodules Ery5, Ery5-TE and Ery6 as well as bimodular DEBS3. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), utilizing native chain Pik chain-elongation intermediates, and dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles, and suggest that substrate recognition has evolved unique characteristics in each system. Additional work has been put forth to establish a general methodology to access DEBS and Pik penta- and hexaketide analogues that encompass a number of stereochemical, structural and functional group variations.

[发布机构] University of Michigan

[效力级别] Chemistry

[关键词] Modular Polyketide Synthases;Chemistry;Science;Chemistry