Structure and Engineering of Natural Product Cytochrome P450 Enzymes

描述(申请人提供)：细胞色素P450是自然界中分布最广泛的一类酶，催化多种天然产物和外来小分子的氧化。虽然已经研究了数百种P450羟基酶在外源物质和类固醇氧化代谢中的作用，但在细菌次生代谢，特别是大环内酯类抗生素生物合成途径中的研究还很少。在大多数这些途径中，羟化(S)发生在生物合成的后期，在聚酮合成酶(PKS)形成大环内酯之后。除了显著提高生物效力外，羟基化还为化学修饰和进一步增强抗感染活性提供了潜在的位点。因此，通过组合生物合成和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广泛底物专一性的大环内酯单加氧酶。这项拟议工作的目的是彻底了解细胞色素P450-PiKC大环内酯单加氧酶的底物灵活性和功能。我们最近对该酶的结构测定为其催化机理和通过12元环大环内酯YC-17和14元环大环内酯纳波霉素羟化生成几个产物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努力更好地了解酶的功能和位置特异性，以及它催化羟化或环氧化反应的能力。此外，我们计划研究史无前例的脱糖胺糖介导的大环内酯类化合物在PIKC结合区域内的锚定，以开发具有多种底物选择性的工程化单加氧酶。具体目标1.纳波霉素与YC-17羟化反应的pikc结构及反应机理的测定(S)。具体目的2.探索大环内酯糖介导的锚定对底物结合和PIKC催化活性的影响。具体目标3.新型大环内酯羟基酶的工程。

DESCRIPTION (provided by applicant): Cytochrome P450s are one of the most widely distributed classes of enzymes in nature, catalyzing the oxidation of a broad range of natural product and xenobiotic small molecules. Although hundreds of P450 hydroxylases 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 pathways, hydroxylation(s) occurs in the late stages of biosynthesis after formation of the macrolide by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of anti-infective activity. Thus, the creation of novel macrolide analogs through combinatorial biosynthesis and chemoenzymatic synthesis warrants a concomitant effort towards the development of macrolide monooxygenases with broad substrate specificity. The aim of the proposed work is to develop a thorough understanding of the substrate flexibility and functionality of the cytochrome P450-PikC macrolide monooxygenase. Our recent structure determination of the enzyme has provided fascinating new insights into its catalytic mechanism and ability to generate several products by hydroxylation of the 12-membered ring macrolide YC-17 and the 14-membered ring macrolide narbomycin. This information will direct protein engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze hydroxylation or epoxidation reactions. Moreover, we plan to investigate the unprecedented desosamine sugar-mediated anchoring of macrolides within the PikC binding domain to develop engineered monooxygenases with versatile substrate selectivity. Specific Aim 1. Determination of the PikC structure and the mechanism(s) of hydroxylation of narbomycin and YC-17. Specific Aim 2. Explore the role of macrolide sugar-mediated anchoring on substrate binding and catalytic activity of PikC. Specific Aim 3. Engineering of novel macrolide hydroxylases.