Structure and Engineering of Natural Product Cytochrome P450 Enzymes

描述（由申请人提供）：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异源小分子的氧化。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种P450羟化酶，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羟化酶。在这些途径中的大多数中，羟基化发生在通过聚酮合酶（PKS）形成大环内酯之后的生物合成的后期阶段。除了显著增加生物效力外，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位点。因此，通过组合生物合成和化学酶促合成产生新的大环内酯类似物，保证了同时努力开发具有广泛底物特异性的大环内酯单加氧酶。拟议的工作的目的是发展的底物的灵活性和功能的细胞色素P450-PikC大环内酯单加氧酶的透彻理解。我们最近的酶的结构测定提供了迷人的新的见解，其催化机制和能力，以产生几个产品的羟基化的12元环大环内酯YC-17和14元环大环内酯那博霉素。这些信息将指导蛋白质工程的努力，以更好地了解酶的功能和位置特异性，以及其催化羟基化或环氧化反应的能力。此外，我们计划研究PikC结合结构域内前所未有的大环内酯类的desosamine糖介导的锚定，以开发具有多功能底物选择性的工程单加氧酶。具体目标1。PikC结构的确定以及纳博霉素和YC-17的羟基化机制。具体目标2。探索大环内酯糖介导的锚定对PikC底物结合和催化活性的作用。具体目标3。新型大环内酯羟化酶的工程化。

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.