Novel Strategies in Light-driven P450 Enzymes

项目总结： 细胞色素P450是血红素硫酸盐单加氧酶的一个超家族，几乎在所有生物中都有发现， 在内源化合物的生物合成和生物降解中发挥重要作用的物质。在人类身上， P450是参与药物代谢和生物活性的主要酶，占总酶的75% 新陈代谢。利用这些单加氧酶的合成潜力也导致了最近对 P450酶在各种化合物后期功能化中的作用。我们实验室已经开发出一种高效的 利用Ru(II)-二亚胺光化学性质的光驱动P450杂化酶系统 光敏剂共价连接到其具有战略定位的非天然单半胱氨酸突变体上 在可见光激发下执行P450反应的血红素结构域。我们最近开始调查一个 具有过氧酶活性的P450酶的互补双分子激活方法是目前研究的热点 目标1。使用各种蛋白质工程方法，我们正在组装一个巨大而多样化的光文库- 引发的P450酶对一系列底物和人具有独特的光催化活性 P450样活动。我们最近还专注于通过结婚来开发化学酶方法 化学催化的优势与P450生物催化的选择性。我们确定了一个很有希望的 底物锚定方法提高P450酶的催化活性并改变其区域选择性。 在目标2中，我们将利用这些策略来合成一个联苯烷酸和 二苯胺类化合物，作为潜在的双重脂肪酸酰胺水解酶(FAAH)/环氧合酶(COX) 用于减轻胃损害的抑制剂，目前困扰着非类固醇抗炎药的使用 (非甾体抗炎药)。计算机对接模拟和生化分析的组合将被用来确定 它们的抑制活性。在目标3中，我们将应用基板工程方法中确定的概念来 为后期衬底功能化开发新平台。 我们的长期目标是利用光驱动的P450酶和化学酶相结合的库 以及开发新药和铅的后期多样化的底物工程战略 化合物。

Project Summary: 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. Harnessing the synthetic potential of these monooxygenases has also led to the recent use of P450 enzymes in late-stage functionalization of various compounds. Our laboratory has developed an efficient light-driven P450 hybrid enzyme system that utilizes the photochemical properties of Ru(II)-diimine photosensitizers covalently attached to strategically positioned non-native single cysteine mutants of their heme domain to perform P450 reactions upon visible light excitation. We recently started to investigate a complementary bimolecular approach to activate P450 enzymes with peroxygenase activity, which is the focus of Aim 1. Using various protein engineering approaches, we are assembling a large and diverse library of light- initiated P450 enzymes with unique photocatalytic activity towards a range of substrates and with human P450-like activities. We have also recently focused on developing chemoenzymatic approaches by marrying the advantages of chemical catalysis with the selectivity of the P450 biocatalysis. We identified a promising substrate anchoring approach to enhance the catalytic activity of P450 enzymes and alter their regioselectivity. In Aim 2, we will capitalize on those strategies to synthesize a library of biphenylalkanoic acid and diphenylamine derivatives, as potential dual fatty acid amide hydrolase (FAAH)/cyclooxygenase (COX) inhibitors to mitigate gastric damages, currently plaguing the use of nonsteroidal anti-inflammatory drugs (NSAIDs). A combination of computer docking simulation and biochemical assays will be used to ascertain their inhibitory activity. In Aim 3, we will apply concepts identified in the substrate engineering approach to develop a new platform for late stage substrate functionalization. Our long-term goal is to employ the libraries of light-driven P450 enzymes combined with the chemoenzymatic and substrate engineering strategies for the development of novel drugs and late stage diversification of lead compounds.