Heme-Dependent Chemistry in Tyrosine Oxidation

酪氨酸氧化中血红素依赖性化学

• 批准号: 10000928
• 负责人: Aimin Liu
• 金额: $ 34.67万
• 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/10000928
• 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; 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; Pathway interactions; Peptides; Peroxidases; Pharmaceutical Preparations; Phenols; Play; Porphyrins; Positioning Attribute; Process; Production; Prosthesis; Protein Engineering; Proteins; Protons; Reaction; 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; trait

PROJECT DESCRIPTION A distinguishing trait of heme enzymes is that a high-valent iron-oxo species is a common oxidant for mediating a remarkable array of oxidation reactions. However, one conundrum is that each enzyme in general promotes only a specific type of reaction. How the reaction type is determined after the formation of the key oxidant remains an open question whose answers have implications for our fundamental understanding of enzyme catalysis as well as de novo enzyme design and protein engineering. Because tyrosine is an important building block of natural products, this application 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 a more common hydroxylation reaction. We found that SfmD is a new member of the tryptophan dioxygenase superfamily that promotes regioselective monooxygenation of 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, the inevitable question arises regarding the governing factors that determine the catalytic activity of these enzymes. In Aim #1, we will determine 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 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 is focused 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 hypotheses. 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.

项目说明 血红素酶的一个显著特征是，高价铁氧物种是一种常见的氧化剂，用于调节 一系列引人注目的氧化反应。然而，一个难题是，每种酶通常只促进一种 特定类型的反应。在关键氧化剂形成后如何确定反应类型仍是一个未知数 这个问题的答案对我们对酶催化的基本理解以及从头开始都有影响 酶设计和蛋白质工程。由于酪氨酸是天然产品的重要组成部分，这 应用重点是三种依赖于血红素的酪氨酸氧化酶的机制特征。每一个 这些酶使用单核血红素辅因子来氧化其酪氨酸底物。有趣的是，一个 结核分枝杆菌细胞色素P450蛋白CyP121催化一种不寻常的氧化碳 交叉偶联反应是比较常见的羟基化反应。我们发现SfmD是一个新的成员 色氨酸双加氧酶超家族，促进甲基化酪氨酸底物的区域选择性单加氧。 过氧化物酶LMBB2与组氨酸而不是半胱氨酸的轴向配体进行过氧酶类型的反应。 这些酶不仅催化基于酪氨酸的氧化反应，而且还与抗菌药物有关 发展。考虑到以血红素为基础的氧化剂和底物结构的相似性，不可避免的 关于决定这些酶的催化活性的控制因素出现了问题。在目标1中，我们 将决定CYP121的机制和结构特征。使用光谱和结构的电池 结合合成探针，我们将揭示一种新的碳-碳偶联机制，该机制由 P450酶。在目标#2中，我们将描述SfmD的结构和机制，重点是如何 底物定位于铁结合的氧化剂和催化中间体的捕获。我们已经确定了 这种蛋白是一种新的基于血红素的加氧酶。目的#3重点研究过氧化物酶催化的反应。 由负责L-3，4-二羟基苯丙氨酸(L-多巴)通过L-酪氨酸形成的LmbB2 羟化作用。我们将利用小分子探测器来询问机械假说。深入分析了 这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产生和 针对芳香底物，揭开表面上看起来的血红素酶的结构-功能关系 不相关的超家族在更高的水平上，并开发潜在的机制，进一步帮助合理的药物设计和 发现过程。