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: 8017192
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8017192
• 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; 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. PUBLIC HEALTH RELEVANCE: This proposal aims at developing a novel biocatalyst to selectively insert an oxygen atom into a substrate C-H bond using visible light and water, under inert atmosphere. This system will be used as an innovative model of human P450 enzymes, which are responsible for the bioactivation and biodegradation of 75% of all drugs.

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，在几乎所有活生物体中发现，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反应是将衍生自分子氧的氧原子引入底物未活性炭中心。该机制涉及铁中心的O-O键的还原性断裂，导致形成高度氧化的血红素自由基铁基物质，即化合物I。大多数P450单加氧酶的特征在于低活性、有限的稳定性、需要昂贵的辅因子（NAD（P）H）来还原血红素铁以及通常依赖于称为还原酶的辅助电子载体蛋白。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氧原子的唯一来源，在惰性气氛下，对各种底物C-H键进行选择性羟基化。该系统将被用作人类P450模型，以促进在药物开发过程的早期鉴定毒性代谢物，并通过生成广泛的羟基化衍生物使先导化合物多样化。该提案的创新部分是用共价连接到血红素结构域的光敏剂Ru（II）二亚胺复合物取代还原酶。然后，在惰性气氛下，通过光氧化途径，直接从酶的静止状态下与Fe（III）结合的水产生反应性化合物I，而不是通过当前的还原机制。这种方法将使得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而导致的蛋白质的快速失活。所提出的系统将导致对底物C-H键的选择性羟基化的酶的活性增强。 公共卫生相关性：本发明的目的在于开发一种新型的生物催化剂，在惰性气氛下，使用可见光和水将氧原子选择性地插入到底物C-H键中。该系统将用作人类P450酶的创新模型，这些酶负责75%药物的生物活化和生物降解。