Enzyme Environmental Effects in Complex Cytochrome P450-Catalyzed Reactions

复杂细胞色素 P450 催化反应中的酶环境影响

• 批准号: 8537211
• 负责人: John C Hackett
• 金额: $ 27.5万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537211
• 关键词: Active Sites; Anabolism; Antifungal Agents; Aromatase; Attention; Azoles; Biological Factors; Biological Models; Biomechanics; CYP19A1 gene; Camphor 5-Monooxygenase; Catalysis; Cessation of life; Chemicals; Chemistry; Cleaved cell; Complex; Computing Methodologies; Coupled; Cytochrome P450; Cytochromes; Data; Development; Disease; Drug Interactions; Drug Targeting; Eicosanoids; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Evaluation; Feedback; Generations; Goals; Health; Hormones; Human; Hybrids; Iron; Kinetics; Lead; Leishmaniasis; Life; Light; Malignant Neoplasms; Mediating; Metabolic Diseases; Metabolism; Methods; Modeling; Mycobacterium tuberculosis; Organic Chemicals; Oxidants; Oxygen; Parasites; Pathway interactions; Pharmaceutical Preparations; Potential Energy; Protons; Public Health; Quantum Mechanics; Raman Spectrum Analysis; Reaction; Resolution; Role; Site-Directed Mutagenesis; Spectrum Analysis; Steroid 17-alpha-monooxygenase; Sterols; Structure-Activity Relationship; Surface; System; Techniques; Temperature; Therapeutic; Xenobiotics; base; chemical synthesis; cryogenics; drug metabolism; electronic structure; flexibility; hormone biosynthesis; infectious disease treatment; inhibitor/antagonist; insight; meetings; molecular dynamics; molecular mechanics; mutant; mycobacterial; novel therapeutics; oxidation; pathogen; pathogenic bacteria; public health relevance; quantum; reactive oxygen intermediate; research study; simulation; small molecule; steroid hormone; theories; therapeutic development

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.

描述（由申请人提供）：细胞色素P450酶（CYP）对于许多天然产物、类固醇激素和类花生酸的生物合成以及大多数药物的清除至关重要。由于它们在异生物质处置中的中心作用，CYP介导许多具有治疗意义的不良药物相互作用。催化O2活化和底物氧化的机制一直具有挑战性，在很大程度上是因为中间体的反应性。切割C-C键的CYP是此类酶中最具机械灵活性的酶;然而，通常没有意识到这组CYP的途径和反应中间体尚未被广泛研究。大多数关于CYP的研究主要集中在羟基化CYP上。因此，对使用多种氧化剂并催化更复杂的转化的酶的关注相对较少。因此，目前可用的实验数据通常不能外推到C-C键裂解CYP。结核分枝杆菌CYP 51是研究O2活化和C-C键断裂机制的一个有价值的典型例子。此外，许多分枝杆菌、锥虫和真菌病原体在其自身的生物合成途径中利用键裂解CYP，其中每一种都是药物靶标。鉴于这些病原体每年造成数百万人死亡，因此非常需要对这些特定酶进行更清晰的机理理解，以支持具有广泛公共卫生重要性的治疗剂的开发。该项目的长期目标是了解C-C键裂解CYP的催化机制，并回答围绕这些酶如何调节其推定的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），第一个具体目标的目标是探索M.结核菌CYP 51激活O2并进行底物氧化。第二个具体目标的目的是验证计算衍生的结构-功能关系，控制活性氧中间体的寿命。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UV-vis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低温辐射分解和共振拉曼光谱来表征相关的C-C键裂解机制的中间体。总而言之，这三个特定目标之间的相互作用将提供理论和实验之间的反馈循环，允许逐步完善机制假设，以提供对CYP酶中活性氧中间体化学的更完整的了解，这对人类健康具有重要影响。 公共卫生相关性： 细胞色素P450是最普遍存在的酶之一，在人体内催化激素生物合成中的几种反应，并在外源物质的代谢中起主导作用。此外，致病菌中的键裂解生物合成细胞色素正在成为治疗感染性疾病的药物靶标。了解这些生物合成酶的结构-功能关系和机制将为开发新的治疗方法和了解整个细胞色素P450酶超家族的机制提供重要的见解。