Structural and functional studies of natural product biosynthetic enzymes

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Natural products are small molecules produced by various organisms and they play a pivotal role in modern drug discovery. Research on bacterial genomes suggests that ~99% of molecules from the global consortium of microbial producers remain to be discovered. Since more and more NP biosynthetic enzymes catalyzing novel and unusual reactions are discovered, there are ample opportunities for new mechanistic insights to be gained from the structural and functional studies of these enzymes. My lab aims to develop a vigorous program in the structural and functional studies of natural product biosynthetic enzymes. In this research proposal, we focus on the enzymes involved in the biosynthesis of polycylic tetramate macrolatams (PoTeMs), an emerging family of bacterial metabolites with a broad spectrum of biological activities. The extraordinary simplicity of the biosynthetic pathway of these compounds is in sharp contrast to their structural complexity. Within the next five years, we will decipher the molecular mechanisms of the NADPH-dependent dehydrogenases IkaC, PtmC and OX4 that govern the inner-ring formation of PoTeMs and elucidate the structural determinants of the P450 enzymes IkaD and CftA that tailor the PoTeM backbone using protein X-ray crystallography as the primary tool, in combination with enzymatic assays of wild-type and mutant proteins. We will also attempt to change the substrate specificity of some of these enzymes via protein engineering. Through these approaches, we aim to elucidate the underlying logic how PoTeMs are furnished and tailored. This study will provide molecular insights into the assembly of PoTeMs and facilitate knowledge-based protein engineering or combinatorial biosynthesis approach to alter or broaden the substrate profile of these enzymes, paving the way for producing novel PoTeMs with improved or new biological activities.