Heme-dependent chemistry in tyrosine oxidation: Diversity Supplement on 5R01GM108988-08 for supporting Samuel Montoya

酪氨酸氧化中的血红素依赖性化学：5R01GM108988-08 上的多样性补充，用于支持 Samuel Montoya

• 批准号: 10167195
• 负责人: Aimin Liu
• 金额: $ 6.11万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10167195
• 关键词: Active Sites; Amino Acid Sequence; Anabolism; Antibiotics; Bacterial Infections; Biological; Carbon; Catalysis; Cations; Characteristics; Charge; Chemicals; Chemistry; Coupled; Coupling; Crystallization; Cysteine; Cytochrome P450; Cytochrome a; Data; Dioxygenases; Distal; Dopa; Drug Design; Electrons; Environment; Enzymes; Funding; Goals; Grant; Health; Heme; Heme Iron; Histidine; Human; Hydrogen Bonding; Hydrogen Peroxide; Hydroxylation; Investigation; Iron; Knowledge; Laboratories; Lead; Levodopa; Ligands; Malignant Neoplasms; Mediating; Molecular; Mononuclear; Mycobacterium tuberculosis; Natural Products; Oxidants; Oxides; Oxygenases; Parents; Pathway interactions; Peptides; Peroxidases; Pharmaceutical Preparations; Phenols; Play; Porphyrins; Positioning Attribute; Process; Production; Prosthesis; Protein Engineering; Proteins; Protons; Reaction; Research Project Grants; Role; Scheme; Structure; Structure-Activity Relationship; System; Testing; Tryptophan; Tuberculosis; Tyrosine; Tyrosine 3-Monooxygenase; antimicrobial drug; antineoplastic antibiotics; antitumor agent; base; c new; cofactor; design; drug development; drug discovery; enzyme activity; enzyme mechanism; heme a; insight; member; metalloenzyme; novel; novel therapeutics; oxidation; pathogen; pathogenic bacteria; small molecule

Abstract of the Parent Research Project One of the hallmarks of heme iron-dependent enzymes is the wide array of oxidation reactions that it can catalyze with its high-valent iron-oxo species. However, one conundrum is that each protein, in general, promotes only a specific type of reaction. How the reaction type is determined after the formation of the critical oxidant remains an open question whose answers have implications for the fundamental understanding of enzyme catalysis as well as de novo enzyme design and protein engineering. Because tyrosine is an essential building block of natural products, this project focuses on a mechanistic characterization of three heme-dependent tyrosine-oxidizing enzymes. Each of these enzymes employs a mononuclear heme cofactor to oxidize its tyrosine-based substrate. Intriguingly, a cytochrome P450 protein, CYP121 from Mycobacterium tuberculosis, catalyzes an unusual oxidative carbon-carbon cross- coupling reaction instead of a more common hydroxylation reaction. We found that SfmD is a new member of the tryptophan dioxygenase superfamily that promotes a regioselective monooxygenation on a methylated tyrosine substrate. The peroxidase LmbB2 performs a peroxygenase-type of reaction with an axial ligand of histidine instead of cysteine. These enzymes not only catalyze tyrosine-based oxidation reactions, but they are also related to antimicrobial drug development. Given the similarities of the heme- based oxidant and the structure of the substrates, an inevitable question arises and will be answered by this study regarding the governing factors that determine the catalytic activity of these enzymes. In Aim #1, we will identify the mechanistic and structural characteristics of CYP121. Using a battery of spectroscopic and structural approaches coupled with synthetic probes, we will unveil a novel carbon- carbon coupling mechanism mediated by the P450 enzyme. In Aim #2, we will characterize the structure and mechanism of SfmD with an emphasis on how the substrate is positioned to the iron-bound oxidant and the capture of catalytic intermediates. We have already identified that this protein is a novel heme- based oxygenase. Aim #3 will focus on studying the peroxidase reaction catalyzed by LmbB2 that is responsible for L-3,4-dihydroxyphenylalanine (L-DOPA) formation through L-tyrosine hydroxylation. We will utilize small-molecule probes to interrogate mechanistic pathways. The in-depth analysis of these three related catalytic systems will test our hypothesis regarding how the heme-bound oxidant is generated and directed to the aromatic substrates, unravel the structure-function relationships of the heme enzymes of seemingly unrelated superfamilies at a higher level, and develop underlying mechanisms further aiding rational drug design and discovery processes.

家长研究项目摘要 依赖于血红素铁的酶的特征之一是它可以进行广泛的氧化反应 用它的高价铁氧物种催化。然而，有一个难题是，总的来说，每种蛋白质 仅促进特定类型的反应。反应类型是如何在形成后确定的 关键氧化剂仍然是一个悬而未决的问题，它的答案对基本的 了解酶催化、从头酶设计和蛋白质工程。因为 酪氨酸是天然产物的重要组成部分，本项目重点介绍了一种 三种依赖于血红素的酪氨酸氧化酶的特性。这些酶中的每一个都利用 一种单核血红素辅因子，用于氧化其酪氨酸底物。有趣的是，一种细胞色素P450 来自结核分枝杆菌的蛋白质CYP121催化一种不寻常的氧化碳-碳交叉反应. 偶联反应而不是更常见的羟基化反应。我们发现SfmD是一种新的 色氨酸双加氧酶超家族的成员，它促进区域选择性的单加氧酶 甲基化酪氨酸底物。过氧化物酶LmbB2执行过氧化物酶类型的反应 取代半胱氨酸的组氨酸的轴向配体。这些酶不仅催化基于酪氨酸的氧化 反应，但它们也与抗菌药物的开发有关。考虑到亚铁血红素的相似性- 基于氧化剂和底物的结构，一个不可避免的问题会出现，并将由 这项关于决定这些酶的催化活性的控制因素的研究。在AIM #1，我们将确定CYP121的机制和结构特征。使用一块电池 光谱和结构方法与合成探针相结合，我们将揭开一种新型的碳- P450酶介导的碳偶联机制。在目标2中，我们将描述结构的特征 以及SfmD的机理，重点是底物如何定位到铁结合的氧化剂上 以及催化中间体的捕获。我们已经确定这种蛋白质是一种新的血红素- 碱性加氧酶。目标#3将重点研究LmbB2催化的过氧化物酶反应，即 负责L-3，4-二羟基苯丙氨酸(L-多巴)通过L-酪氨酸羟基化生成。我们 将利用小分子探测器来询问机械路径。对这些问题的深入分析 三个相关的催化系统将检验我们关于血红素结合的氧化剂是如何 生成并定向到芳香底物，解开结构与功能的关系 看似不相关的超家族的血红素酶在更高的水平上，并发展潜在的 进一步帮助合理的药物设计和发现过程的机制。