Heme-Dependent Chemistry in Aromatic Oxidation

芳香族氧化中血红素依赖性化学

基本信息

批准号：
10540085
负责人：
Aimin Liu
金额：
$ 32.35万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10540085
关键词：
Acids Address Affect Amino Acids Anabolism Antibiotics Antineoplastic Agents Aromatic Compounds Biochemistry Biogenesis Biological Biological Process Catabolism Catalysis Cations Chemicals Chemistry Crystallization Crystallography Development Dioxygenases Distal Drug Design Electron Spin Resonance Spectroscopy Environment Enzymes Family Funding Generations Genetic Goals Health Heme Heme Group Hemeproteins Histidine Human Hydrogen Peroxide Hydroxylation In Situ Isotope Labeling Kinetics Knowledge Lead Levodopa Mediating Metabolism Mixed Function Oxygenases Molecular Monitor Natural Products Outcome Oxidants Oxidation-Reduction Oxides Oxygen Oxygenases Pathway interactions Pharmacologic Substance Play Porphyrins Process Property Protein Engineering Proteins Quinones Reaction Regulation Roentgen Rays Role Rotation Site Source Specificity Spectrum Analysis Structure System Temperature Testing Time Tryptophan Tyrosine Work analog anti-cancer antimicrobial drug base catalyst chemical reaction cofactor design dopaquinone drug development drug discovery enzyme mechanism heme a insight member millisecond natural antimicrobial novel oxidation oxindole pyrroline recruit scaffold thioether x-ray free-electron laser

项目摘要

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.

芳香族氧化中血红素依赖性化学抽象的含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。组氨酸连接的血红素酶，例如血红素依赖性芳香加氧酶 (HDAO) 超家族，可以使用分子氧或过氧化氢来氧化其底物。什么目前尚不清楚产生这些酶及其各自的分子因素特异性，即在产生关键活性氧化剂后，如何催化单一反应类型。这这个问题的答案将拓宽我们对酶催化、从头酶的基本理解设计和蛋白质工程。该应用侧重于结构和机械特性 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 是一组依赖血红素形成的左旋多巴 (L-DOPA) 具有吡咯啉部分的抗生素生物合成中的单加氧酶。 SfmD 介导区域选择性 3-甲基-L-酪氨酸的羟基化用于合成 Saframycin A 的核心醌结构。 MarE 形式许多化合物的羟吲哚结构。这些酶在生物合成途径中发挥着重要作用在许多具有生物活性的天然产物和药物先导化合物中发现了次生代谢物。我们的主要目标是了解控制其羟基化机制的关键因素及其用这些酶形成氧化中间体的不同策略。我们利用了广泛的使用非规范氨基酸探测这些酶机制的复杂分子细节的方法酸、底物类似物、时间分辨紫外可见光谱和 EPR 光谱、动力学和同位素标记研究，我们已经成功地将路径上的反应中间体捕获在晶体中。拟议的工作将测试我们的假设：i) 血红素基氧化剂是如何产生的，ii) 氧气是如何被引导到底物，以及iii)揭示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，以帮助支架的合理开发药物设计和发现过程。