Heme-Dependent Chemistry in Tyrosine 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。是什么 目前尚不清楚产生这些酶的分子因素及其各自的特异性， 也就是说，在生成关键的活性氧化剂之后，如何催化单一的反应类型？这个问题的答案是 这个问题将扩大我们对酶催化、从头开始的酶设计和蛋白质的基本理解。 工程学。本应用重点研究了三种芳香族化合物的结构和机理表征。 参与天然产物和抗菌药物生物合成的HDAO超家族的氧化酶， 包括具有吡咯啉部分或核心醌结构的抗生素。这些酶在体内起着至关重要的作用 次生代谢物的生物合成途径存在于许多生物活性天然产物和 药物先导化合物。我们的主要目标是了解支配他们的 羟化机理及其与这些酶形成氧化中间体的不同策略。我们 已经利用广泛的方法来探索这些酶的复杂的分子细节 使用非规范氨基酸、底物类似物、时间分辨UV-Vis和EPR光谱的机理， 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)基于血红素的氧化剂如何 二)氧是如何被导向底物的，以及三)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。

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 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.