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.

项目描述 血红素酶的一个区别特征是，高价值铁氧化物种是介导A的常见氧化剂 出色的氧化反应。但是，一个难题是通常每种酶仅促进 特定类型的反应类型。在钥匙氧化剂的形成后如何确定反应类型保持开放 问题的答案对我们对酶催化的基本理解以及从头开始有影响 酶设计和蛋白质工程。因为酪氨酸是天然产品的重要组成部分，所以 应用侧重于三种血红素依赖性酪氨酸氧化酶的机械表征。每个 这些酶采用单核血红素辅因子来氧化其基于酪氨酸的底物。有趣的是， 细胞色素P450蛋白，结核分枝杆菌的CYP121，催化不寻常的氧化碳碳 交叉偶联反应改为更常见的羟基化反应。我们发现SFMD是 色氨酸二氧酶超家族，可促进甲基化酪氨酸底物的区域选择性单氧化。 过氧化物酶LMBB2用组氨酸而不是半胱氨酸的轴向配体进行反应的过氧酶类型。 这些酶不仅催化酪氨酸基氧化反应，还与抗菌药物有关 发展。鉴于血红素基氧化剂的相似性和底物的结构，不可避免 关于决定这些酶催化活性的治疗因素的问题。在AIM＃1中，我们 将确定CYP121的机械和结构特征。使用光谱和结构的电池 方法与合成探针结合，我们将推出一种新型的碳碳耦合机制，由该机理介导 P450酶。在AIM＃2中，我们将表征SFMD的结构和机制，重点是如何 底物位于结合的氧化剂和催化中间体的捕获上。我们已经确定了 该蛋白是一种新型的基于血红素的氧合酶。 AIM＃3专注于研究过氧化物酶反应催化的 由LMBB2通过L-二苯胺（L-DOPA）形成LMBB2。 羟基化。我们将利用小分子探针来审问机械假设。深入分析 这三个相关的催化系统将检验我们关于如何产生血红素结合氧化剂的假设，并 针对芳族底物，揭示了看似在 在更高级别上无关的超家族，发展潜在的机制进一步帮助理性药物设计和 发现过程。