铁氧还蛋白调控P450催化活性的结构机制研究

Cytochrome P450 monooxygenases (P450s) are capable of catalyzing the selective oxidation reactions under mild conditions that are highly challenging in organic synthesis, thus holding great application potential in natural product biosynthesis and drug precursor screening. Ferreoxins (Fdxs), as the required electron shuttle vectors, are one of the key factors affecting the catalytic efficiency of P450s. However, it remains unclear how a P450 enzyme and an Fdx match each other, and what are the structural basis for the molecular recognition between P450s and Fdxs. To answer these important questions, in this project, we chose to study the P450 enzyme PikC and its endogenous svFdxs from Streptomyces venezuelae in order to clarify the above mentioned mechanism. First, we plan to analyze the effects of different svFdxs on the catalytic activity of PikC in vitro. Second, we will solve the three-dimensional X-ray crystal structures of a number of svFdxs that differentially regulate the PikC activity. Based on structural and biochemical results, the structural mechanism for the regulation of P450 activity by ferredoxins will be elucidated. Furthermore, this study will provide new insights into improvement of P450 catalytic efficiency and promote its industrial applications.

细胞色素P450单加氧酶能够在温和条件下催化许多在有机合成中极具挑战性的选择性氧化反应，在天然产物生物合成及药物前体筛选中有很大的应用前景。铁氧还蛋白（ferredoxin，Fdx）作为P450活性必须的电子穿梭载体是影响P450催化效率的重要因素，但Fdx如何与P450酶选择配对？Fdx通过何种机制影响P450酶催化效率尚不清楚。为了阐明这些科学问题，本项目拟选择委内瑞拉链霉菌中的P450酶PikC及其内源性svFdx为研究对象，利用体外酶反应重构分析不同svFdx对PikC催化活性的影响；运用结构生物学方法解析对PikC活性产生差异化影响的svFdx的三维结构，通过结构分析和生化分析阐明svFdx影响PikC催化效率及产物分布的结构机制，为提高P450酶的催化效率、促进其工业应用提供新的思路和理论基础。

细胞色素P450单加氧酶能够在温和条件下催化许多在有机合成中极具挑战性的选择性氧化反应，在天然产物生物合成及药物前体筛选中有很大的应用前景。铁氧还蛋白（ferredoxin，Fdx）、铁氧还蛋白还原酶（Ferredoxin reductase， FdR）作为I型P450酶活性必须的电子穿梭载体是影响P450催化效率的重要因素，这些还原伴侣组合是通过何种机制影响P450酶催化效率尚不清楚。为了阐明这些科学问题，本项目以本课题组发现的Fdx1499为电子传递中间体分别与一系列不同的FdR进行组合完成了对P450sca2催化的体外酶反应重构；解析了3个不同FdR的晶体结构; 通过结构分析和生化分析阐明FdR影响电子传递和催化效率的关键机制，为提高P450酶的催化效率、促进其工业应用提供新的思路和理论基础。