Structural and Biochemical Investigation of Substrate Recognition and Shuttling in Natural Product Biosynthesis.

[摘要] Polyketide natural products are chemically complex, bioactive small molecules with multiple therapeutic applications. Modular polyketide synthases (PKSs), which biosynthesize polyketides, are organized into assembly lines with modules that successively extend and in some cases modify specific pathway intermediates. Throughout biosynthesis an acyl carrier domain (ACP) tethers and transports intermediates to each catalytic domain within its module then transfers the fully processed intermediate to the next module. Employing x-ray crystallography, electron cryo-microscopy (cryo EM), and biochemical experiments, this thesis investigates the substrate and ACP specificity of PKS catalytic domains, the architecture of a module, dynamics of the catalytic domains and the ACP during a catalytic cycle, and the mechanisms of substrate transfer between modules.A PKS β-module is comprised of a ketosynthase (KS), an acyltransferase (AT), and a ketoreductase (KR), which elongate (KS and AT) and modify (KR) the polyketide intermediate. Crystal structures of a KS-AT di-domain and a KR domain along with structural comparison to homologs provided insights into the molecular basis of catalysis and substrate specificity. Cryo EM provided the first structure of an entire PKS β-module. The architecture of the PKS β-module forms a single chamber, in which the intramodular ACP can access all active sites. Incubation with different combinations of the natural substrates allowed visualization of the ACP at distinct positions during the catalytic cycle. The cryo-EM reconstructions and crystal structures revealed the ACP docking site on each catalytic domain and important protein-protein interactions that were validated by biochemical experiments. Docking domains promote modular association and intermediate transfer between modules. Structural and biochemical analysis identified a Class 2 docking domain from cyanobacterial PKSs with a novel docking strategy for promoting intermediate chain transfer. Through structural and biochemical analysis we also identified a thioesterase domain, which appears to function in intermediate transfer by releasing a pathway intermediate from an ACP for transfer to the next module in the pathway. The information presented in this thesis provides an important advance in our understanding of the PKS biosynthetic machinery and new engineering tools that will facilitate bioengineering efforts to produce novel small molecules with desired therapeutic activity.

[发布机构] University of Michigan

[效力级别] Polyketide Biosynthesis

[关键词] Natural Product Biosynthesis;Polyketide Biosynthesis;Structural Biology;Biological Chemistry;Science;Chemical Biology