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

 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，在几乎所有的活生物体中发现，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反应是将来自分子氧的氧原子选择性地引入到底物未活化的碳中心。该机制涉及两个还原当量的消耗和血红素中心的分子氧的活化，导致形成高度氧化的卟啉自由基铁基物质，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）以及通常依赖于称为还原酶的辅助电子载体蛋白。我们已经开发了一种有效的光驱动的P450 BM 3杂合酶系统，该系统避免了使用还原酶和昂贵的NADPH辅因子。这种方法利用钌（II）光敏剂的光化学性质共价连接到战略定位的非天然的单一半胱氨酸残基的P450 BM 3血红素结构域突变体能够进行P450反应后，光活化。优化的混合P450 BM 3酶获得的高光催化活性和初始反应速率验证了我们的光活化方法，并为当前的提议奠定了基础。我们正计划解决高活性杂合酶（目标1）的晶体结构，以了解控制有效电子转移的因素，以及改善未来杂合酶的设计。我们还将利用杂交酶的独特性质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（目的2）。最后两个目标将致力于使用能够产生各种人类药物代谢物的P450 BM 3突变体（目标3）和使用已知羟基化大体积疏水性异生物质的哺乳动物P450酶CYP 2B 4（目标4）来扩大光激活方法的范围。这种光激活P450酶方法将被用作人类P450模型，以促进在药物开发过程的早期鉴定毒性代谢物，并通过产生广泛的羟基化衍生物使先导化合物多样化。

 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.