Biosynthesis of Amino Acid Derived Quinone Cofactors

氨基酸衍生的醌辅因子的生物合成

• 批准号: 7587378
• 负责人: CAROLINE MARY WILMOT
• 金额: $ 27.72万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7587378
• 关键词: 6-hydroxydopa; Aerobic; Aldehydes; Alzheimer' s Disease; Amines; Amino Acids; Anabolism; Anti-Inflammatory Agents; Anti-inflammatory; Bacteria; Behavior; Biogenesis; Biology; Carcinogens; Catalysis; Chemicals; Chemistry; Complex; Complications of Diabetes Mellitus; Congestive; Copper; Cytochrome P450; Cytochrome c Peroxidase; Enzymes; Evolution; Family; Fat-Soluble Vitamin; Fatty Acids; Freezing; Genes; Grant; Heart Diseases; Heme; Herbicides; Human; Hydrogen Peroxide; Hypoxia; Inflammatory Response; Insecticides; Ions; Life; Link; Metabolic; Metals; Microspectrophotometry; Molecular; Oxidation-Reduction; Oxidative Stress; Oxygen; Paracoccus denitrificans; Peroxidases; Pharmaceutical Preparations; Pichia; Play; Post-Translational Protein Processing; Process; Property; Proteins; Quinones; Reaction; Roentgen Rays; Role; Steroids; Structure; Trimethylamine-N-oxide reductase; Tryptophan; X-Ray Crystallography; Yeasts; aminoacid biosynthesis; base; cofactor; diamino oxhydrase; diheme cytochrome c; drug metabolism; fatty acid biosynthesis; man; metal complex; methylamine dehydrogenase; mutant; novel; polypeptide; therapeutic target

DESCRIPTION (provided by applicant): Found in all kingdoms of life, copper-containing amine oxidases (CuAOs) contain two redox centers. One is a copper ion, and the other is a novel organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), derived from the post-translational modification of a Tyr residue in the protein. The biosynthesis of TPQ is an auto-catalytic process, which is molecular oxygen and cupric ion dependent. Formation of such self- processed cofactors likely played a key role in enzyme evolution, as they extended the chemical functionalities available for catalysis without the evolution of separate biosynthetic enzymes. Methylamine dehydrogenase (MADH), a metabolic enzyme found in methylotrophic/autotrophic bacteria, also contains a quinone cofactor, tryptophan tryptophylquinone (TTQ), derived from the post-translational modification of two Trp residues in the protein. In contrast to CuAOs, the maturation of MADH involves at least 4 other proteins. In the previous grant period, we have begun to characterize one of these proteins, MauG. It is a highly unusual diheme enzyme responsible for the completion of TTQ synthesis, which can use either molecular oxygen or hydrogen peroxide as a substrate. In the last few years, the list of enzymes containing amino acid derived cofactors has grown rapidly. In addition, both TPQ and TTQ synthesis require oxygen substrates, but in very different reactions. The ability to activate molecular oxygen underpins all aerobic biology, but the details of how this is achieved are still poorly understood. I propose to use the yeast Hansenula polymorpha methylamine oxidase (HPAO) to study the structural basis of TPQ formation. Through a novel combination of single crystal UV/visible microspectrophotometry, X-ray crystallography and anaerobic / aerobic freeze trapping, reaction intermediates in biogenesis will be trapped in the crystal. The controversial role of copper in CuAO will be explored through metal replacement studies. In addition, I propose to explore MADH maturation through X-ray crystallographic studies of MauG, in particular focusing on the atypical behavior of the c-type heme(s).PUBLIC HEALTH RELEVANCE: The aberrant actions of human copper-containing amine oxidases are linked to congestive heart disease, late-diabetic complications and Alzheimer's disease. One human copper-containing amine oxidase is involved in the inflammatory response, and represents a new target for anti-inflammatory drugs. MauG is sequentially related to peroxidases that detoxify H2O2 under hypoxic conditions (oxidative stress), but unusually can also activate molecular oxygen with mechanistic similarities to human cytochrome P450 enzymes, the major players in drug metabolism, carcinogen activation, biosynthesis of physiologically important molecules, such as steroids, fat-soluble vitamins and fatty acids, as well as the degradation of insecticides and herbicides.

描述（由申请人提供）：在所有生命界中发现，含铜胺氧化酶（CuAO）含有两个氧化还原中心。一个是铜离子，另一个是一种新的有机辅因子，2，4，5-三羟基苯丙氨酸醌（TPQ），来自蛋白质中Tyr残基的翻译后修饰。TPQ的生物合成是一个依赖于分子氧和铜离子的自催化过程。这种自我加工的辅因子的形成可能在酶进化中发挥了关键作用，因为它们扩展了可用于催化的化学功能，而无需进化单独的生物合成酶。甲胺脱氢酶（MADH）是一种在甲基营养/自养细菌中发现的代谢酶，也含有一种醌辅因子，色氨酸甲萘醌（TTQ），来源于蛋白质中两个Trp残基的翻译后修饰。与CuAO相反，MADH的成熟涉及至少4种其他蛋白质。在上一个赠款期间，我们已经开始表征这些蛋白质之一，MauG。它是一种非常不寻常的二血红素酶，负责完成TTQ合成，可以使用分子氧或过氧化氢作为底物。在过去的几年中，含有氨基酸衍生的辅因子的酶的列表迅速增长。此外，TPQ和TTQ合成都需要氧底物，但在非常不同的反应中。激活分子氧的能力是所有有氧生物学的基础，但如何实现这一点的细节仍然知之甚少。我建议使用酵母多形汉逊酵母甲胺氧化酶（HPAO）研究TPQ形成的结构基础。通过单晶紫外/可见显微分光光度法，X射线晶体学和厌氧/好氧冷冻捕集的新组合，生物发生中的反应中间体将被捕获在晶体中。铜在CuAO中有争议的作用将通过金属置换研究来探讨。此外，我建议探索MADH成熟通过X-射线晶体学研究的MauG，特别是专注于非典型行为的C-型血红素（S）。公共卫生相关性：人类含铜胺氧化酶的异常行动与充血性心脏病，晚期糖尿病并发症和阿尔茨海默氏病。一种人类含铜胺氧化酶参与炎症反应，并代表抗炎药物的新靶点。MauG依次与在缺氧条件下（氧化应激）解毒H2 O2的过氧化物酶相关，但不寻常地也可以激活分子氧，其机制与人类细胞色素P450酶相似，细胞色素P450酶是药物代谢、致癌物活化、生理学重要分子（如类固醇、脂溶性维生素和脂肪酸）的生物合成以及杀虫剂和除草剂降解的主要参与者。