Heme-Dependent Chemistry in Aromatic Oxidation

家长研究项目摘要 含有血红素的酶无处不在，有助于各种过程，并催化大量的 氧化化学反应。组氨酸连接的亚铁血红素酶，如依赖亚铁血红素的芳香族 加氧酶(HDAO)超家族，可以利用分子氧或过氧化氢来氧化它们的 底物。目前还不清楚的是产生这些酶的分子因素。 它们各自的特性，即在生成关键的活性氧化剂之后，单个反应如何 催化型？这个问题的答案将扩大我们对酶的基本理解。 催化、从头酶设计和蛋白质工程。此应用程序侧重于结构 和HDAO超家族中三种芳香族氧化酶的机理研究 在天然产物和抗微生物药物的生物合成中，包括含有吡咯啉部分或 核心对苯二酚结构。这些酶在次生物质的生物合成途径中起着至关重要的作用。 在许多具有生物活性的天然产品和药物先导化合物中发现的代谢物。我们的 主要目标是了解控制它们的羟基化机制的关键因素和它们的 与这些酶形成氧化中间体的不同策略。我们已经利用了一个广泛的 使用以下方法探索这些酶机制的复杂分子细节的方法 非规范氨基酸，底物类似物，时间分辨UV-Vis和EPR光谱，动力学， 和同位素标记研究，我们已经成功地捕获了路径上的反应 晶体中的中间体。这项拟议的工作将检验我们的假设：i)以血红素为基础的 氧化剂是如何产生的，ii)氧气是如何被导向底物的，以及iii)解开 影响催化作用。拟议研究的结果是对这三个相关问题的深入了解 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.