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: 8325771
• 负责人: John C Hackett
• 金额: $ 4.8万
• 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/8325771
• 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.

描述（由申请人提供）：细胞色素P450酶（CYPS）对于多种天然产物，类固醇激素和类eicosanoids的生物合成以及大多数药物的清除至关重要。由于其在异种生物处置中的核心作用，CYP介导了许多具有治疗意义的不良药物相互作用。 CYP催化的O2激活和底物氧化的机制在很大程度上是挑战，这在很大程度上是由于中间体的反应性。裂解C-C键的CYP是此类酶的最灵活的CYP。但是，通常尚未意识到，这组CYP的途径和反应性中间体尚未得到广泛研究。大多数关于CYP的研究主要集中在羟基CYPS上。因此，对使用多种氧化剂并催化更复杂的转化的CYP酶的关注很少。因此，目前可用的实验数据通常不能被外推到C-C键切割CYP。结核分枝杆菌CYP51构成了研究O2激活和C-C键裂解机制的有价值且典型的例子。此外，许多分枝杆菌，锥虫和真菌病原体都利用键在自己的生物合成途径中切割CYP，每种CYP是一种药物靶标。鉴于这些病原体每年造成数百万死亡，因此对这些特定酶的理解非常清楚，以支持开发广泛的公共卫生重要性的治疗学。该项目的长期目标是了解C-C键切割CYP的催化机制，并回答有关这些酶如何调节其假定氧中间体的反应性的问题。采用分子动力学模拟和杂交量子力学/分子力学技术（QM/mm），第一个特定目的的目的是探索结核分枝杆菌CYP51使用的几种可能的反应机制来激活O2并执行底物氧化。第二个特定目的的目的是验证计算衍生的结构功能关系，该关系控制了活性氧中间体的寿命。为了满足这些目标，将利用催化中间体，定向诱变，停止流量的紫外线和共振拉曼技术的有机化学合成。在计算结果的指导下，第三个特定目的的目的是使用冷冻溶解和共振拉曼光谱表征相关的CYP中间体，以阐明C-C键裂解机制。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反馈回路，从而使机械假设的逐步完善，以更完整地了解CYP酶中的活性氧中间化学，对人类健康具有重要意义。 公共卫生相关性： 细胞色素P450是最普遍的酶之一，在人类中，催化了激素生物合成中的几种反应，并且在异物代谢中具有主要作用。此外，致病细菌中的粘结裂解生物合成细胞色素正在成为治疗传染病的药物靶标。了解这些生物合成酶的结构功能关系和机制将为开发新疗法的发展以及对整个细胞色素P450酶的机制的理解提供重要的见解。