Structure and Engineering of Natural Product Cyctochrome P450 Enzymes

描述(由申请人提供)：我们寻求更新一项高效的多PI研究计划，该计划涉及从不同的天然产物途径中分析和设计广泛类别的单加氧酶。细胞色素P450是自然界分布最广泛的一组酶，催化天然产物和外源小分子的氧化。虽然已经研究了数百个P450在外源化合物和类固醇的氧化代谢中的作用，但在细菌次级代谢，特别是大环内酯类抗生素的生物合成途径中的研究还很少。在大多数这些体系中，羟基化和/或环氧化反应发生在聚酮合成酶(PKS)形成大环内酯类化合物后的生物合成的后期阶段。除了显著提高生物效力外，羟化还为化学修饰和进一步增强生物活性提供了潜在的位点。因此，通过体内代谢工程和体外化学酶合成来创造新的大环内酯类似物，需要同时努力开发具有特定底物特异性的单加氧酶。这项拟议工作的目的是扩大我们对大环内酯类P450单加氧酶底物的灵活性和功能性的了解，并选择其他天然产物体系。我们在第一期支撑期的进展为这些生物催化剂的分子机制提供了有趣的新见解，以及它们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。这些信息将指导蛋白质工程/底物工程的努力，以更好地了解酶的功能和位置特异性，以及它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来创建不同的底物，生物化学来研究和开发具有多种底物选择性的工程单加氧酶，以及基于X射线和核磁共振的方法来获得高分辨率的结构信息，以从机制上理解这些非凡的蛋白质。具体目标1.使用一系列天然大环内酯底物YC-17的合成类似物，评估空间、电子和定向基团因素对P450 pikc催化混杂的影响。使用基于X射线和溶液核磁共振的结构生物学方法，获得对结合参数、蛋白质-底物动力学以及天然和非天然底物区域和立体化学特异性的机制基础的详细见解。具体目标2.为一系列新的P450酶扩大获得不同合成底物的途径，以研究单加氧酶催化的羟化和环氧化反应的区域和立体化学细节。具体目标3.开展混合功能重复P450酶的生化和结构研究，以分析底物专一性和动力学，以及探索结合和催化机制。

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.