Enzyme Environmental Effects in Complex Cytochrome P450-Catalyzed Reactions

描述（由申请人提供）：细胞色素 P450 酶 (CYP) 对于许多天然产物、类固醇激素和类二十烷酸的生物合成以及大多数药物的清除至关重要。由于其在异生素处置中的核心作用，CYP 介导许多具有治疗意义的不良药物相互作用。 CYP 催化 O2 活化和底物氧化的机制一直难以阐明，这在很大程度上是因为中间体的反应性。裂解 C-C 键的 CYP 是此类酶中机械性最灵活的酶之一。然而，人们通常没有意识到这组 CYP 的途径和反应中间体尚未得到广泛研究。大多数 CYP 的研究主要集中在羟基化 CYP 上。因此，对于使用多种氧化剂并催化更复杂转化的 CYP 酶，人们的关注相对较少。因此，目前可用的实验数据不能普遍推断为 C-C 键裂解 CYP。结核分枝杆菌 CYP51 为研究 O2 活化和 C-C 键裂解机制提供了一个有价值的原型实例。此外，许多分枝杆菌、锥虫和真菌病原体在它们自己的生物合成途径中利用键断裂的 CYP，每个途径都是药物靶点。鉴于这些病原体每年导致数百万人死亡，因此迫切需要对这些特定酶有更清晰的机制了解，以支持开发具有广泛公共卫生重要性的疗法。该项目的长期目标是了解 C-C 键裂解 CYP 的催化机制，并回答有关这些酶如何调整其假定氧中间体的反应性的问题。应用分子动力学模拟和混合量子力学/分子力学技术（QM/MM），第一个特定目标的目标是探索结核分枝杆菌CYP51激活O2并进行底物氧化的几种可能的反应机制。第二个具体目标的目标是验证计算得出的控制活性氧中间体寿命的结构-功能关系。为了实现这些目标，将利用催化中间体的有机化学合成、定点诱变、停流紫外-可见光和共振拉曼技术。在计算结果的指导下，第三个具体目标的目标是使用冷冻放射分解和共振拉曼光谱来表征相关的 CYP 中间体，以揭示 C-C 键断裂机制。总而言之，这三个具体目标之间的相互作用将提供理论和实验之间的反馈循环，从而逐步完善机制假设，从而更全面地了解 CYP 酶中的活性氧中间体化学，这对人类健康具有重要意义。 公共卫生相关性： 细胞色素 P450 是最普遍存在的酶之一，在人体中催化激素生物合成中的多种反应，并在外来物质的代谢中起主导作用。此外，病原菌中的键断裂生物合成细胞色素正在成为治疗传染病的药物靶点。了解这些生物合成酶的结构功能关系和机制将为开发新疗法和理解整个细胞色素 P450 酶超家族的机制提供重要的见解。

DESCRIPTION (provided by applicant): The cytochrome P450 enzymes (CYPs) are essential for the biosynthesis of numerous natural products, steroid hormones, and eicosanoids, as well as the clearance of most drugs. Due to their central role in xenobiotic disposition, CYPs mediate many adverse drug interactions of therapeutic significance. The mechanisms of CYP catalyzed O2 activation and substrate oxidation have been challenging to unravel, in large part because of the reactivity of intermediates. The CYPs that cleave C-C bonds are among the most mechanistically flexible of such enzymes; however, it is not usually realized that the pathways and reactive intermediates of this group of CYPs have not yet been investigated extensively. Most studies on CYP have been primarily focused on the hydroxylating CYPs. Thus relatively little attention has been paid to the CYP enzymes which use multiple oxidants and catalyse the more complicated transformations. Thus the presently available experimental data cannot be generally extrapolated to the C-C bond cleaving CYPs. Mycobacterium tuberculosis CYP51 constitutes a valuable and prototypical example for the study of O2 activation and C-C bond cleavage mechanisms. Moreover, many mycobacterial, trypanosomal, and fungal pathogens utilize bond cleaving CYPs in their own biosynthetic pathways, each of which is a drug target. Given that these pathogens are responsible for millions of deaths annually, there is a profound need for a clearer mechanistic understanding of these particular enzymes in support of the development of therapeutics of broad public health importance. The long-term goal of this project is to understand the catalytic mechanisms of C-C bond cleaving CYPs, and to answer questions surrounding how these enzymes tune the reactivity of their putative oxygen intermediates. Applying molecular dynamics simulation and hybrid quantum mechanics/molecular mechanics techniques (QM/MM), the objective of the first Specific Aim is to explore the several possible reaction mechanisms utilized by M. tuberculosis CYP51 to activate O2 and perform substrate oxidation. The objective of the second Specific Aim is to validate the computationally-derived structure-function relationships governing the lifetimes of reactive oxygen intermediates. To meet these objectives, organic chemical syntheses of catalytic intermediates, site-directed mutagenesis, stopped-flow UV-vis, and resonance Raman techniques will be utilized. Guided by computational results, the objective of the third Specific Aim is to characterize relevant CYP intermediates using cryoradiolysis and resonance Raman spectroscopy to shed light on the C-C bond cleavage mechanism. Taken together, the interplay between these three Specific Aims will provide a feedback loop between theory and experiment, allowing incremental refinement of mechanistic hypotheses to provide a more complete understanding of reactive oxygen intermediate chemistry in CYP enzymes with important implications for human health. PUBLIC HEALTH RELEVANCE: The cytochromes P450 are among the most ubiquitous enzymes, and in humans, catalyze several reactions in hormone biosynthesis and have a dominant role in the metabolism of foreign substances. Furthermore, bond cleaving biosynthetic cytochromes in pathogenic bacteria are emerging as drug targets for the treatment of infectious diseases. Understanding the structure-function relationships and the mechanisms of these biosynthetic enzymes will provide important insight towards the development of new therapeutics and the understanding of mechanisms of the entire cytochrome P450 enzyme superfamily.