Structure and Engineering of Natural Product Cyctochrome P450 Enzymes

描述（由申请人提供）：我们寻求更新一个高生产力的多PI研究计划，涉及从不同的天然产物途径的单加氧酶的广泛类别的分析和工程。细胞色素P450是自然界中分布最广泛的一类酶，催化天然产物和外源性小分子的氧化。虽然已经在外源性物质和类固醇的氧化代谢中检测了数百个P450，但在细菌次级代谢中，特别是在大环内酯类抗生素生物合成途径中，仅研究了少数P450。在大多数这些系统中，羟基化和/或环氧化反应发生在生物合成的后期阶段，在通过聚酮合酶（PKS）形成大环内酯之后。除了显著增加生物学效力外，羟基化还为化学修饰和进一步增强生物活性提供了潜在位点。因此，通过体内代谢工程和体外化学酶促合成产生新的大环内酯类似物，保证了对具有确定的底物特异性的单加氧酶的开发的伴随努力。拟议的工作的目的是扩大我们的理解的底物的灵活性和功能的范围P450单加氧酶从大环内酯和选择其他天然产物系统。我们在第一个支持期内取得的进展为这些生物催化剂的分子机制及其通过羟基化和天然和非天然底物的环氧化产生新产品的能力提供了令人着迷的新见解。这些信息将指导蛋白质工程/底物工程工作，以更好地了解酶的功能和位置特异性，以及其催化一系列氧化反应的能力。我们的计划带来了合成化学的互补方法，以创建不同的底物，生物化学研究和开发具有多功能底物选择性的工程单加氧酶，以及基于X射线和NMR的方法，以获得高分辨率的结构信息，用于对这些显着蛋白质的机械理解。具体目标1。使用天然大环内酯底物YC-17的一系列合成类似物评估空间、电子和导向基团因素对P450 PikC中催化混杂性的影响。采用基于X射线和溶液NMR的结构生物学方法，详细了解结合参数、蛋白质-底物动力学以及天然和非天然底物的区域和立体化学特异性的机理基础。具体目标2。扩大对一系列新型P450酶的不同合成底物的使用，以研究单加氧酶催化的羟基化和环氧化反应的区域和立体化学细节。具体目标3。进行混合功能迭代P450酶的生物化学和结构研究，以分析底物特异性和动力学，并研究结合和催化机制。

DESCRIPTION (provided by applicant): We seek renewal of a highly productive multiple-PI research program involving the analysis and engineering of a broad class of monooxygenases from diverse natural product pathways. Cytochrome P450s are one of the most widely distributed groups of enzymes in nature, catalyzing the oxidation of natural product and xenobiotic small molecules. Although hundreds of P450s have been examined in the oxidative metabolism of xenobiotics and steroids, only a small number have been studied in bacterial secondary metabolism, especially in macrolide antibiotic biosynthetic pathways. In most of these systems, hydroxylation and/or epoxidation reactions occur in the late stages of biosynthesis after macrolide formation by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of bioactivities. Thus, the creation of novel macrolide analogs through in vivo metabolic engineering and in vitro chemoenzymatic synthesis warrants a concomitant effort towards the development of monooxygenases with defined substrate specificities. The aim of the proposed work is to expand our understanding of the substrate flexibility and functionality of a range of P450 monooxygenases from macrolide and select other natural product systems. Our progress over the first period of support has provided fascinating new insights into the molecular mechanisms of these biocatalysts, and their ability to generate novel products by hydroxylation, and epoxidation of natural and unnatural substrates. This information will direct protein engineering/substrate engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze a range of oxidative reactions. Our program brings complementary approaches of synthetic chemistry to create diverse substrates, biochemistry to investigate and develop engineered monoxygenases with versatile substrate selectivity, and X-ray and NMR- based methods to obtain high resolution structural information for mechanistic understanding of these remarkable proteins. Specific Aim 1. Assess the impact of steric, electronic and directing group factors on catalytic promiscuity in the P450 PikC using a series of synthetic analogs of the natural macrolide substrate YC-17. Employ X-ray and solution NMR based structural biology approaches to gain detailed insights into binding parameters, protein-substrate dynamics, and the mechanistic basis for regio- and stereochemical specificity of natural and unnatural substrates. Specific Aim 2. Expand access to diverse synthetic substrates for a range of new P450 enzymes to investigate regio- and stereochemical details of monooxygenase-catalyzed hydroxylation and epoxidation reactions. Specific Aim 3. Pursue biochemical and structural studies of mixed-function iterative P450 enzymes to analyze substrate specificity and kinetics, as well as to investigate binding and catalytic mechanisms.