Structural and functional studies of natural product biosynthetic enzymes

天然产物是由横跨所有生命王国的大量有机体产生的。这些小分子及其衍生物广泛用于日常生活，也是人类药物的主要来源。近年来，越来越多的微生物基因组测序和基因组学指导的方法发现了许多新的天然产物，有力地标志着发现新的生物活性天然产物的第二个黄金时代的开始。大自然进化出了一系列令人眼花缭乱的酶来合成这些天然产品。许多天然产物复杂的化学结构和引人入胜的功能往往是有趣和不寻常的酶催化的结果。我提出了一个关于天然产物生物合成中新的和重要的酶的结构和功能研究的计划，这将提供对天然产品生物合成潜在逻辑的机械洞察。这将有可能极大地促进我们对各种生物合成途径的理解，并为通过理性或组合方法产生非天然天然产物提供新的重新设计视角。我们的研究将包括酶及其与底物、产物及其类似物的络合物的X射线结晶学研究，以及使用定点突变和酶分析进行的功能研究。我们将把所学到的知识应用于设计具有改变特性的新酶。在本申请所涵盖的时间段，我将重点介绍细菌生物碱生物合成途径中的新型酶。吲哚倍半萜(IST)主要存在于植物中，是含吲哚生物碱的天然产物。直到最近才在原核生物中发现了ISTs。这些分子具有稀有的五元环系统，显示出有趣的生物活性。我们的具体目标是了解夏霉素/奥西霉素生物合成途径中的两个新的黄素依赖加氧酶HAXI和HAYK是如何催化细菌ISTS生物合成的异常反应的。这项研究将促进蛋白质工程改变这些酶的底物分布，为产生具有改进或新的生物活性的新生物碱铺平道路。

Natural products are produced by an abundance of organisms spanning all kingdoms of life. These small molecules and their derivatives are widely used in everyday living and also constitute a major source of human medicine. In recent years, sequencing of more and more microbial genomes and the genomics-guided approaches have uncovered numerous novel natural products, strongly indicating the beginning of the second golden age for the discovery of new bioactive natural products. Nature has evolved a dazzling array of enzymes to synthesize these natural products. The chemically complex structure and fascinating function of many natural products is often the result of interesting and unusual enzymatic catalysis. I propose a program on structural and functional studies of the novel and important enzymes in natural product biosynthesis which will provide mechanistic insight into the underlying logic of natural product biosynthesis. This will have potential to significantly advance our understanding of various biosynthetic pathways and offer new redesign perspectives for the generation of unnatural natural products by rational or combinatorial approaches. Our research will involve X-ray crystallographic studies of enzymes and their complexes with substrates, products and their analogues combined with functional studies using site-directed mutagenesis and enzymatic assays. We will apply acquired knowledge toward engineering new enzymes with altered specificities. In the period covered by this application, I will focus on novel enzymes in bacterial alkaloid biosynthetic pathways. Indolosesquiterpenes (ISTs), primarily found in plants, are indole-containing alkaloid natural products. Only very recently were ISTs found in prokaryotes. These molecules featuring rare pentacylic ring systems display interesting biological activities. Our specific objective is to understand how XiaI and XiaK, two novel flavin-dependent oxygenases in the xiamycin/oxiamycin biosynthetic pathway, catalyze the unusual reactions for bacterial ISTs biosynthesis. This study will facilitate protein engineering to alter the substrate profile of these enzymes, paving the way for generating novel alkaloids with improved or new biological activities.**