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: 8625311
• 负责人: Lionel E. Cheruzel
• 金额: $ 10.76万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8625311
• 关键词: Accounting; Acetaminophen; Active Sites; Adrenergic beta-Antagonists; Anabolism; Binding; Biodegradation; Camphor; Camphor 5-Monooxygenase; Carbon; Carrier Proteins; Complex; Coughing; Cysteine; Cytochrome P450; Dependence; Dextromethorphan; Electron Transport; Electrons; Enzymes; Gas Chromatography; Generations; Goals; Heme; Heme Iron; Human; Hydrogen Bonding; Hydroxylation; Iron; Label; Lead; Libraries; Life; Light; Medicine; Metabolism; Mixed Function Oxygenases; Modeling; Modification; Molecular; Organism; Oxidoreductase; Oxygen; Pathway interactions; Pharmaceutical Preparations; Photosensitizing Agents; Play; Positioning Attribute; Process; Property; Propranolol; Proteins; Provider; Publishing; Reaction; Reactive Oxygen Species; Relative (related person); Research; Rest; Role; Route; Site-Directed Mutagenesis; Source; System; Terpenes; Visible Radiation; Water; cofactor; cytochrome P-450 CYP119 (Sulfolobus solfataricus); drug development; drug metabolism; enzyme activity; improved; innovation; irradiation; leukotoxin; limonene; long chain fatty acid; mutant; novel; novel strategies; oxidation; planetary Atmosphere; public health relevance; scale up; thermophilic organism

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 introduction of an oxygen atom, derived from molecular oxygen, into a substrate unactivated carbon center. The mechanism involves the reductive scission of the O-O bond at the iron center, leading to the formation of a highly oxidative heme radical ferryl species, namely Compound I. Most P450 monooxygenases are characterized by low activity, limited stability, need of an expensive cofactor (NAD(P)H) to reduce the heme iron and general dependence on auxiliary electron carrier proteins called reductases. We propose to develop a novel P450 system that will utilize light and water as the only source of oxygen atom to perform, under inert atmosphere, the selective hydroxylation of various substrate C-H bonds. This system 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. The innovative part of this proposal is to replace the reductase by a photosensitizer Ru(II) diimine complex covalently attached to the heme domain. The reactive Compound I will then be generated, under inert atmosphere, via a photo-oxidative route directly from the water bound to the Fe(III) in the resting state of the enzyme rather than through the current reductive mechanism. This approach will enable the control of the electron flow between the photosensitizer and the heme domain while avoiding the rapid deactivation of the protein due to reactive oxygen species. The proposed system will result in enhanced activity of the enzymes towards the selective hydroxylation of substrate C-H bonds.

描述（由申请人提供）：细胞色素P450是几乎所有活体中发现的血红素 - 硫代硫酸硫酸盐单加氧酶的超家族，它们在内源性化合物的生物合成和生物降解中起着重要作用。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反应是引入源自分子氧衍生的氧原子，降为底物未激活的碳中心。该机制涉及在铁中心的O-O键的还原性分裂，从而导致形成高度氧化的血红素自由基轮渡物种，即复合I。大多数P450单加仑酶的特征是低活性，有限的稳定性，需求有限，需要昂贵的辅助因子（ NAD（p）H）减少了血红素铁和对称为还原酶的辅助电子载体蛋白的普遍依赖性。我们建议开发一种新型的P450系统，该系统将利用光和水作为惰性大气下执行各种底物C-H键的选择性羟基化的唯一氧原子来源。该系统将被用作人类P450模型，以促进药物开发过程中早期有毒代谢产物的鉴定，并通过产生广泛的羟基化衍生物，使铅化合物的多样化能够多样化。该提案的创新部分是用光敏剂RU（II）二胺复合物取代了血红素结构域的光敏剂RU（II）二胺复合物。然后，在惰性气氛下，我将通过光氧化途径直接从酶的静止状态而不是通过当前的还原机制来生成反应性化合物。这种方法将能够控制光敏剂和血红素结构域之间的电子流，同时避免由于活性氧引起的蛋白质快速失活。所提出的系统将导致酶对底物C-H键选择性羟基化的活性增强。