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: 8322837
• 负责人: John C Hackett
• 金额: $ 28.52万
• 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/8322837
• 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 Delivery Systems; Drug Interactions; 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.

说明(申请人提供)：细胞色素P450酶(Cyps)是许多天然产物、类固醇激素和二十烷类化合物的生物合成所必需的，也是大多数药物清除所必需的。由于其在异种生物处置中的核心作用，它介导了许多具有治疗意义的药物不良相互作用。CYP催化的O2活化和底物氧化的机制一直是一个具有挑战性的机制，很大程度上是由于中间体的反应性。裂解C-C键的Cyps是这类酶中最灵活的一种；然而，人们通常没有意识到这类Cyps的途径和活性中间体还没有得到广泛的研究。目前对细胞色素P的研究主要集中在羟基化的细胞色素上。因此，对于使用多种氧化剂并催化更复杂的转化的CYP酶的研究相对较少。因此，目前可用的实验数据一般不能外推到C-C键断裂的Cyps。结核分枝杆菌CYP51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它们自己的生物合成途径中利用键断裂的Cyps，每一条途径都是药物靶标。鉴于这些病原体每年造成数百万人死亡，因此迫切需要对这些特定的酶有更明确的机械理解，以支持具有广泛公共卫生重要性的治疗方法的发展。该项目的长期目标是了解C-C键断裂Cyps的催化机制，并回答围绕这些酶如何调节其假定的氧中间体的反应活性的问题。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和执行底物氧化的几种可能的反应机制。第二个特定目标的目标是验证通过计算得出的结构-功能关系，该关系支配着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，第三个特定目标的目标是利用低温辐射和共振拉曼光谱来表征相关的CYP中间体，以揭示C-C键断裂的机理。综上所述，这三个特定目标之间的相互作用将在理论和实验之间提供一个反馈回路，允许逐步完善机械假设，以提供对CYP酶中的活性氧中间化学的更完整的理解，对人类健康具有重要的影响。 公共卫生相关性： 细胞色素P450是最普遍存在的酶之一，在人类体内，催化激素生物合成的几个反应，并在外来物质的新陈代谢中起主导作用。此外，病原菌中断裂键的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，将为开发新的治疗药物和理解整个细胞色素P450酶超家族的机制提供重要的见解。