Biosynthesis of Amino Acid Derived Quinone Cofactors

描述(申请人提供)：发现于所有的生命王国，含铜的胺氧化酶(CuAO)含有两个氧化还原中心。一种是铜离子，另一种是一种新的有机辅因子2，4，5-三羟基苯丙氨酸醌(TPQ)，它是由蛋白质中Tyr残基的翻译后修饰而来的。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。这种自处理辅因子的形成可能在酶的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，而不需要单独的生物合成酶的进化。甲胺脱氢酶(MADH)是一种存在于甲基营养细菌/自养细菌中的代谢酶，它还含有一种由蛋白质中两个色氨酸残基翻译后修饰而来的苯醌辅因子色氨酸色氨酸。与CuAOS不同的是，MADH的成熟至少涉及4种其他蛋白质。在之前的赠款期间，我们已经开始对其中一种蛋白质--MAUG进行表征。它是一种非常不寻常的双氢酶，负责完成TTQ的合成，它可以利用分子氧或过氧化氢作为底物。在过去的几年里，含有氨基酸衍生辅因子的酶的名单迅速增长。此外，TPQ和TTQ的合成都需要氧气底物，但反应方式截然不同。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)来研究TPQ形成的结构基础。通过单晶UV/可见光显微分光光度、X射线结晶学和厌氧/好氧冷冻捕获的新组合，生物发生中的反应中间体将被捕获到晶体中。铜在Cuao中的有争议的作用将通过金属替代研究来探索。此外，我建议通过对MAUG的X射线结晶学研究来探索MADH的成熟，特别是关注c型血红素(S)的非典型行为。PUBLLIC健康相关性：人类含铜胺氧化酶的异常活动与充血性心脏病、晚期糖尿病并发症和阿尔茨海默病有关。一种人类含铜胺氧化酶参与炎症反应，代表了抗炎药物的新靶点。MAUG依次与过氧化物酶相关，后者在低氧条件下(氧化应激)解毒过氧化氢，但也异常地激活分子氧，其机制与人类细胞色素P450酶相似，后者是药物代谢、致癌物质激活、类固醇、脂溶维生素和脂肪酸等重要生理分子的生物合成以及杀虫剂和除草剂降解的主要参与者。

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.