Heme-Dependent Chemistry in Aromatic Oxidation

芳香族氧化中依赖于血红素的化学 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。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，可以使用分子氧或过氧化氢来氧化其底物。什么 目前还不清楚产生这些酶的分子因素和它们各自的 特殊性，即在产生关键的活性氧化剂之后，如何催化单一反应类型。这个 回答这个问题将扩大我们对酶催化的基本认识，从头开始酶 设计和蛋白质工程。本应用侧重于结构和机械特性的表征 HDAO超家族的三种芳香族氧化酶，参与天然产物和 抗菌药物生物合成：TyrH、SfmD和Mare。TyrH是一组依赖于血红素的L-多巴形成 含有吡咯啉部分的抗生素生物合成中的单加氧酶。SfmD调节区域选择性 3-甲基-L-酪氨酸羟基化合成红霉素A的核心醌结构 许多化合物的氧化吲哚结构。这些酶在生物合成途径中发挥着重要作用。 在许多生物活性天然产物和药物先导化合物中发现的次生代谢物。 我们的主要目标是了解控制它们的羟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。我们已经利用了广泛的 使用非正则氨基探索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UV-Vis和EPR光谱、动力学和同位素标记研究， 我们已经成功地在晶体中捕获了同路径反应中间体。拟议的工作将 测试我们的假设：i)基于血红素的氧化剂是如何产生的，ii)氧气是如何直接进入 底物，以及iii)解开影响催化的结构因素。建议的研究结果是 深入了解这三种相互关联的催化体系，有助于支架的合理开发 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.