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 血红素铁依赖性酶的标志之一是它可以发生广泛的氧化反应 用其高价铁氧物种进行催化。然而，一个难题是，一般来说，每种蛋白质， 仅促进特定类型的反应。形成后如何确定反应类型 关键氧化剂仍然是一个悬而未决的问题，其答案对基本氧化剂有影响 了解酶催化以及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，该项目重点关注机械 三种血红素依赖性酪氨酸氧化酶的表征。这些酶中的每一种都使用 一种单核血红素辅助因子，可氧化其基于酪氨酸的底物。有趣的是，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是一个新的 色氨酸双加氧酶超家族的成员，促进区域选择性单加氧 甲基化酪氨酸底物。过氧化物酶 LmbB2 与以下物质进行过氧化酶类型的反应： 组氨酸的轴向配体而不是半胱氨酸。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。鉴于血红素的相似性 基于氧化剂和底物的结构，不可避免地会出现一个问题，并将通过以下方式回答 这项研究涉及决定这些酶催化活性的控制因素。瞄准 #1，我们将确定 CYP121 的机械和结构特征。使用电池 光谱和结构方法与合成探针相结合，我们将推出一种新型碳- 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 SfmD 和机制，重点是底物如何定位到铁结合氧化剂 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型血红素- 基于加氧酶。目标 #3 将重点研究 LmbB2 催化的过氧化物酶反应，即 负责通过 L-酪氨酸羟基化形成 L-3,4-二羟基苯丙氨酸 (L-DOPA)。我们 将利用小分子探针来探究机制途径。对这些的深入分析 三个相关的催化系统将检验我们关于血红素结合氧化剂如何作用的假设 生成并定向到芳香底物，揭示了芳香族底物的结构-功能关系 在更高水平上看似不相关的超家族的血红素酶，并发展潜在的 机制进一步帮助合理的药物设计和发现过程。