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：
10681493
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/10681493
关键词：
Acids Affect Amino Acids Anabolism Antibiotics Antineoplastic Agents Aromatic Compounds Binding Biochemistry Biogenesis Biological Biological Process Catabolism Catalysis Cations Chemicals Chemistry 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 Learning Ligation Mediating Metabolism Mixed Function Oxygenases Molecular Monitor Natural Products Outcome Oxidants Oxidation-Reduction 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 Visit 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 secondary metabolite structural determinants 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.

芳族氧化中的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，并催化大量氧化化学。组氨酸绑扎的血红素酶，例如血红素依赖性芳族氧酶（HDAO）超家族可以使用分子氧或过氧化氢来氧化其底物。什么目前尚不清楚的是使这些酶及其各自的分子因素特异性，即在生成关键活性氧化剂后，如何催化单一反应类型。这回答这个问题将扩大我们对酶催化，从头酶的基本理解设计和蛋白质工程。该应用集中于结构和机械表征 HDAO超家族的三种芳族氧化酶与天然产物和抗菌药物生物合成：Tyrh，SFMD和Mare。 Tyrh是一组血红素依赖性的L-DOPA形成吡咯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-取and的羟基化，用于合成Saframycin A. Mare形成的核心喹酮结构许多化合物的氧丝结构。这些酶在生物合成途径中起重要作用在许多生物活性天然产物和药物铅化合物中发现的二级代谢产物。我们的主要目标是了解控制其羟基化机制的关键因素与这些酶形成氧化中间体的不同策略。我们已经利用了使用非规范氨基探测这些酶机制的复杂分子细节的方法酸，底物类似物，时间分辨的UV-VIS和EPR光谱，动力学和同位素标记的研究，而且我们已经成功地捕获了Crystallo中的Pathway反应中间体。拟议的工作将测试我们关于i）如何产生血红素的氧化剂的假设底物和iii）揭示影响催化的结构因素。拟议研究的结果是对这三种相关催化系统的深入了解，以帮助开发脚手架的理性药物设计和发现过程。