Ru(II)-diimine labeled P450 mutants for the selective hydroxylation of substrate

Ru(II)-二亚胺标记的 P450 突变体用于底物的选择性羟基化

• 批准号: 8854787
• 负责人: Lionel E. Cheruzel
• 金额: $ 10.84万
• 依托单位: SAN JOSE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8854787
• 关键词: Accounting; Anabolism; Architecture; Binding; Biodegradation; CYP2B4 gene; Carbon; Carrier Proteins; Communities; Complex; Consumption; Cysteine; Cytochrome P450; Cytochromes; Dependence; Dioxygen; Electron Transport; Electrons; Enzymes; Foundations; Future; Generations; Goals; Heme; High Pressure Liquid Chromatography; Homologous Gene; Human; Hybrids; Hydroxylation; Label; Laboratories; Lauric Acids; Lead; Life; Light; Manuscripts; Metabolism; Mixed Function Oxygenases; Modeling; Molecular; NADP; Organism; Oxidation-Reduction; Oxidoreductase; Oxygen; Pathway interactions; Pharmaceutical Preparations; Photosensitizing Agents; Plasmids; Play; Porphyrins; Positioning Attribute; Process; Property; Protein Isoforms; Proteins; Reaction; Research; Roentgen Rays; Role; Structure; System; TimeLine; Visible Radiation; Xenobiotics; cofactor; design; drug development; drug metabolism; enzyme activity; hybrid enzyme; improved; innovation; instrument; mutant; public health relevance; reaction rate; symposium

 DESCRIPTION (provided by applicant): Cytochromes P450 are a superfamily of heme-thiolate monooxygenases, found in almost all living organisms that play an important role in the biosynthesis and biodegradation of endogenous compounds. In humans, P450s are the major enzymes involved in drug metabolism and bioactivation, accounting for 75% of the total metabolism. The classical P450 reaction is the selective introduction of an oxygen atom, derived from molecular dioxygen, into a substrate unactivated carbon center. The mechanism involves the consumption of two reducing equivalents and the activation of dioxygen at the heme center, leading to the formation of a highly oxidative porphyrin radical ferryl species, namely Compound I. Most P450 monooxygenases are characterized by low activity, limited stability, need of an expensive cofactor (NAD(P)H) and general dependence on auxiliary electron carrier proteins called reductases. We have developed an efficient light-driven P450 BM3 hybrid enzyme system that circumvents the use of the reductase and the expensive NADPH cofactor. This approach utilizes the photochemical properties of Ru(II) photosensitizers covalently attached to strategically positioned non-native single cysteine residues of P450 BM3 heme domain mutants able to perform P450 reactions upon light activation. The high photocatalytic activity and initial reaction rates obtained with the optimized hybrid P450 BM3 enzymes validated our light- activated approach and lays the foundation for the current proposal. We are planning on solving the crystal structure of the highly active hybrid enzyme (Aim 1) in order to understand the factors controlling the efficient electron transfer as well as to improve the design of future hybrd enzymes. We will also take advantage of the unique properties of the hybrid enzymes to study P450 protein interactions thought to play an important role in human enzyme activity (Aim 2). The last two aims will be dedicated to expanding the scope of the light- activated approach using P450 BM3 mutants able to generate various human drug metabolites (Aim 3) and using a mammalian P450 enzyme, CYP2B4, known to hydroxylate bulky hydrophobic xenobiotics (Aim 4). This light-activated P450 enzyme approach will be used as a human P450 model to facilitate the identification of toxic metabolites early in the drug development process and to enable the diversification of lead compounds through the generation of a broad range of hydroxylated derivatives.

 描述（由申请人提供）：细胞色素P450是血红素硫醇单加氧酶的超家族，存在于几乎所有活的生物体中，在内源性化合物的生物合成和生物降解中发挥重要作用。在人体中，P450是参与药物代谢和生物活化的主要酶，占总代谢的75%。经典的 P450 反应是将分子双氧衍生的氧原子选择性引入底物未活性碳中心。该机制涉及两个还原当量的消耗和血红素中心双氧的激活，导致形成高度氧化的卟啉自由基，即化合物 I。大多数 P450 单加氧酶的特点是活性低、稳定性有限、需要昂贵的辅因子 (NAD(P)H) 以及通常依赖于称为还原酶的辅助电子载体蛋白。我们开发了一种高效的光驱动 P450 BM3 混合酶系统，避免使用还原酶和昂贵的 NADPH 辅因子。该方法利用 Ru(II) 光敏剂的光化学特性，该光敏剂共价连接到 P450 BM3 血红素结构域突变体的策略性定位的非天然单个半胱氨酸残基上，能够在光激活时执行 P450 反应。使用优化的混合 P450 BM3 酶获得的高光催化活性和初始反应速率验证了我们的光激活方法，并为当前的提案奠定了基础。我们计划解决高活性杂化酶的晶体结构（目标1），以了解控制有效电子转移的因素并改进未来杂化酶的设计。我们还将利用杂合酶的独特特性来研究 P450 蛋白相互作用，认为它们在人类酶活性中发挥着重要作用（目标 2）。最后两个目标将致力于扩大光激活方法的范围，使用能够产生各种人类药物代谢物的 P450 BM3 突变体（目标 3），并使用哺乳动物 P450 酶 CYP2B4，已知该酶可以羟基化大的疏水性异生物质（目标 4）。这种光激活 P450 酶方法将用作人类 P450 模型，以促进在药物开发过程的早期识别有毒代谢物，并通过生成广泛的羟基化衍生物实现先导化合物的多样化。