. This program investigates the role of protein'protein interactions in carrier protein-dependent biosynthesis, including fatty acid synthase, polyketide synthase, and non-ribosomal peptide synthetase pathways. The natural products associated with these pathways serve as therapeutics including antibiotics, antifungals, and anticancer agents. The ability to direct the biosynthesis of these pathways for the production of untried bioactive compounds is of critical importance for new and improved therapies. In prior years, we demonstrated how protein'protein interactions between carrier proteins and partner protein domains direct reactivity and guide processivity. Moreover, we developed a suite of chemical biology tools to stabilize and interrogate these interactions at atomic resolution through innovative crosslinker development and structural biology. We now propose to expand these tools with new, caged crosslinkers designed to capture transient partner proteins. With this comprehensive library of crosslinkers, we will evaluate carrier protein interactions with partner proteins that include ketoreductases, enoyl reductases, and thioesterases from fatty acid and polyketide synthases, ketosynthase/chain-length factors and acyltransferases from polyketide synthases, and condensation domains, halogenases, and oxidases from non-ribosomal peptide synthetases. Once captured, these crosslinked species will be studied by solution-phase NMR, kinetic and thermodynamic assays, X-ray crystallography, and molecular dynamics simulations to more fully elucidate mechanism and specificity conferred by carrier protein-substrate/intermediate/produ interactions. Finally, we will use data collected in this program as an informatic platform to enable synthetic biological production of new natural product hybrids.
. Natural products provide critical medicines to treat human disease; therefore, an understanding of their biosynthesis will directly support development of new and improved natural product-based therapeutics. Developing on outstanding progress in recent years towards revealing the fundamental protein?protein interactions between biosynthetic enzymes, this proposal aims to further our knowledge through the development of tools to structurally characterize biosynthetic machinery at atomic resolution with temporal acuity. These studies will provide a foundation to rationally manipulate these pathways, affording access to new molecular species for next-generation drug discovery and development.
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