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（由适用提供）：细胞色素P450是血红素硫酸硫酸单加氧酶的超家族，在几乎所有活体中都发现，在内源性化合物的生物合成和生物降解中起重要作用。在人类中，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反应是选择性引入源自分子二氧化物的氧原子中的氧原子，降落到底物未激活的碳中心中。 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 characterised by low activity, limited stability, need of an expensive cofactor (NAD(P)H) and general dependence on auxiliary electron carrier Proteins called还原。我们已经开发了一种有效的轻度驱动P450 BM3混合酶系统，该酶绕过还原和昂贵的NADPH辅因子的使用。这种方法利用Ru（II）光敏剂的光化学特性共价附加到战略性定位的非本地单半胱氨酸保留的P450 BM3血红素结构域突变体可以在光激活后执行P450反应。用优化的杂化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，并为当前的建议奠定了基础。我们计划解决高度活性混合酶的晶体结构（AIM 1），以了解控制有效电子传输的因素，并改善未来混合酶的设计。我们还将利用杂化酶的独特特性来研究被认为在人类酶活性中起重要作用的P450蛋白质相互作用（AIM 2）。最后两个目标将用于使用P450 BM3突变体扩大光激活方法的范围，以生成各种人类药物代谢物（AIM 3），并使用哺乳动物的P450酶，CYP2B4，已知，羟基盐酸盐盐液生物质Xentobiotics已知（AIM 4）。这种光激活的P450酶方法将用作人类P450模型，以促进药物发育过程中有毒代谢物的鉴定，并通过产生广泛的羟基化衍生物来使铅化合物多样化。