The goal of component 2 is to study the specificity of protein-protein interactions and substrate recognition processes involved in the biosynthesis of natural products by applying NMR spectroscopy. The overwhelming majority of antibiotics and chemotherapeutics are natural products, many of which are biosynthesized by Non-Ribosomal Peptide Synthetases (NRPSs). These assembly line protein clusters can reach up to mega-Dalton size. NRPS products include antibiotics, antitumor agents, antiviral and immunosuppressive drugs, and fungal or bacterial toxins. In contrast to ribosomal protein synthesis, little is known about the mechanisms by which NRPSs assemble their products. Structural data on isolated domains become increasingly available but little is known about mechanisms of protein interactions or substrate recognition, which must involve precise recognition of activated substrates, growing chains, and final hydrolysis and release from the assembly line. All substrate recognition and protein interaction processes must be mediated by the terniary structures of the domains and modules of the synthetases, since no coding is available as it is for ribosomal peptide biosynthesis. Basic mechanisms ofthe recognition are widely unknown. We propose to study the processes of domain interactions and the specificity of substrate recognition in this enterobactin synthetase NRPS cluster. The assembly line for the iron chelator enterobactin consists ofthe short one module NRPS EntF, and a split module formed by the isolated enzyme EntE and the di-domainal EntB. Since enterobactin synthetases are widely conserved in enterobacteria they are emerging targets for development of new antibacterial drugs.
Our specific aims are: 1: Structures of holo-EntF T-TE di-domain and ofthe condensation domain EntF C 2: Structure, dynamics and interactions between the EntF A and T domains. 3: Complex formation and dynamic interactions of full-length type I NRPS EntF. 4: Substrate localization and mechanistic studies of the Ent NRPS assembly. 5: Structure-based design of inhibitors to support anti-microbial therapies.

Public Health Relevance

Understanding the structural principles by which substrates are specifically recognized and incorporated may allow the exploration of genetically encoded clusters for the biosynthesis or engineering of new assembly lines for production of novel molecules that could alleviate the emergence of multiple and extreme drug resistance against the majority of established antibiotics including last resort treatments.

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Harvard Medical School
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