Heme-Dependent Chemistry in Aromatic Oxidation

芳香氧化中的血红素依赖化学 摘要 含血红素的酶是普遍存在的，有助于各种过程并催化大量的生物降解。 氧化化学组氨酸连接的血红素酶，如血红素依赖性芳香族加氧酶 （HDAO）超家族，可以使用分子氧或过氧化氢来氧化它们的底物。什么 目前尚不清楚的是产生这些酶的分子因素， 特异性，即，关键活性氧化剂产生后，单一反应类型如何催化。的 这个问题的答案将拓宽我们对酶催化的基本理解，从头酶 设计和蛋白质工程。本申请集中于以下物质的结构和机械表征： HDAO超家族的三种芳香族氧化酶，涉及天然产物， 抗微生物药物生物合成：TyrH、SfmD和MarE。TyrH是一组血红素依赖性L-DOPA形成酶， 单加氧酶在具有吡咯啉部分的抗生素的生物合成中的作用。SfmD介导的区域选择性 3-甲基-L-酪氨酸的羟基化反应，用于合成沙夫霉素A的核心醌结构。MarE表格 许多化合物的羟吲哚结构。这些酶在生物合成途径中起着重要作用。 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次级代谢物。 我们的主要目标是了解控制其羟基化机制的关键因素， 不同的策略与这些酶形成氧化中间体。我们使用了广泛的 使用非规范氨基来探测这些酶机制的复杂分子细节的方法 酸、底物类似物、时间分辨紫外-可见和EPR光谱、动力学和同位素标记研究， 并且我们已经成功地在晶体中捕获途径上的反应中间体。拟议的工作将 测试我们关于i）血红素基氧化剂是如何产生的，ii）氧是如何被引导到 底物，和iii）解开影响催化的结构因素。拟议研究的结果是， 深入了解这三个相关的催化系统，以帮助开发合理的支架， 药物设计和发现过程。

Heme-Dependent Chemistry in Aromatic Oxidation Abstract 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 that are involved in natural product and antimicrobial drug biosynthesis: TyrH, SfmD, and MarE. TyrH is a group of heme-dependent L-DOPA forming monooxygenase in the biosynthesis of antibiotics with a pyrroline moiety. SfmD mediates the regioselective hydroxylation of 3-methyl-L-tyrosine for synthesizing the core quinone structure of saframycin A. MarE forms an oxindole structure for many compounds. These enzymes play important 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 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.