Heme-Dependent Chemistry in Aromatic Oxidation

母研究项目摘要 含血红素的酶是普遍存在的，有助于各种过程和催化大量的 氧化化学反应。组氨酸连接的血红素酶，如血红素依赖性芳香族血红素酶， 加氧酶（HDAO）超家族，可以使用分子氧或过氧化氢来氧化它们的 印刷受体.目前尚不清楚的是产生这些酶的分子因素， 它们各自的特性，即，关键活性氧化剂生成后， 类型催化的？这个问题的答案将拓宽我们对酶的基本理解 催化、从头酶设计和蛋白质工程。该应用程序侧重于结构 三种芳香族氧化酶的HDAO超家族参与的机制表征 在天然产物和抗微生物药物生物合成中，包括具有吡咯啉部分或 核心醌结构。这些酶在次生代谢物的生物合成途径中发挥着重要作用 在许多具有生物活性的天然产物和药物先导化合物中发现的代谢物。我们 主要目标是了解控制其羟基化机制的关键因素及其 不同的策略与这些酶形成氧化中间体。我们利用了广泛的 一系列的方法来探测这些酶机制的复杂分子细节， 非规范氨基酸，底物类似物，时间分辨UV-vis和EPR光谱，动力学， 和同位素标记的研究，我们已经成功地在捕获的途径反应 晶体中间体。拟议的工作将测试我们的假设，关于i）如何基于血红素 氧化剂的产生，ii）氧如何被引导到基底，以及iii）解开 影响催化作用。拟议研究的结果是深入了解这三个相关的 催化系统，以帮助开发合理的药物设计和发现过程的支架。

Abstract of the Parent Research Project Heme-containing enzymes are ubiquitous, contributing to various processes and catalyzing a vast array of oxidative chemistries. Histidine-ligated heme enzymes, such as the heme-dependent aromatic oxygenase (HDAO) superfamily, can use either molecular oxygen or hydrogen peroxide to oxidize their substrates. What is currently not understood are the molecular factors that engender these enzymes with their respective specificities, i.e., after generation of the critical active oxidant, how is a single reaction type catalyzed? The answer to this question will broaden our fundamental understanding of enzyme catalysis, de novo enzyme design, and protein engineering. This application focuses on the structural and mechanistic characterization of three aromatic-oxidizing enzymes of the HDAO superfamily involved in natural product and antimicrobial drug biosynthesis, including antibiotics with a pyrroline moiety or a core quinone structure. These enzymes play essential roles in the biosynthetic pathway of secondary metabolites found in many biologically active natural products and pharmaceutical lead compounds. Our primary goal is to understand the key factors that govern their hydroxylation mechanisms and their differing strategies to form oxidizing intermediates with these enzymes. We have utilized a broad spectrum of approaches to probe the intricate molecular details of these enzyme mechanisms using noncanonical amino acids, substrate analogs, time-resolved UV-vis and EPR spectroscopies, kinetics, and isotope-labeling studies, and we have been successful in trapping on-pathway reaction intermediates in crystallo. The proposed work will test our hypotheses regarding i) how the heme-based oxidant is generated, ii) how oxygen is directed to the substrate, and iii) unravel the structural factors that affect catalysis. The outcome of the proposed studies is an in-depth understanding of these three related catalytic systems to aid the development of scaffolds for rational drug design and discovery processes.