Structural and functional studies of natural product biosynthetic enzymes

天然产物是由各种生物产生的小分子，它们在现代药物发现中起着关键作用。对细菌基因组的研究表明，来自全球微生物生产者联盟的约99%的分子仍有待发现。由于越来越多的NP生物合成酶催化新的和不寻常的反应被发现，有足够的机会，从这些酶的结构和功能的研究获得新的机制的见解。本实验室致力于天然产物生物合成酶的结构和功能研究。在这项研究中，我们专注于参与生物合成的多环四酸大环内酯（PoTeMs），一个新兴的家庭的细菌代谢产物具有广泛的生物活性的酶。这些化合物的生物合成途径非常简单，与它们的结构复杂性形成鲜明对比。在接下来的五年里，我们将破译NADPH依赖性辅酶IkaC，PtmC和OX4的分子机制，这些酶控制PoTeM的内环形成，并阐明P450酶IkaD和CftA的结构决定因素，这些酶使用蛋白质X射线晶体学作为主要工具，结合野生型和突变蛋白的酶促测定来定制PoTeM骨架。我们还将尝试通过蛋白质工程改变这些酶的底物特异性。通过这些方法，我们的目标是阐明底层逻辑如何PoTeMs提供和定制。这项研究将为PoTeMs的组装提供分子见解，并促进基于知识的蛋白质工程或组合生物合成方法来改变或拓宽这些酶的底物谱，为生产具有改进或新的生物活性的新型PoTeMs铺平道路。

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.