Studies on rho-Cresol Methylhydroxylase

对甲酚甲基羟化酶的研究

• 批准号: 6325386
• 负责人: WILLIAM S MCINTIRE
• 金额: $ 28.7万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325386
• 关键词: Escherichia coli; Parkinson's disease; Pseudomonas; X ray crystallography; active sites; apoenzymes; binding sites; computer graphics /printing; covalent bond; electron transport; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; flavin adenine dinucleotide; flavoproteins; fluorimetry; heme; mitochondrial disease /disorder; oxidoreductase; oxygenases; protein purification; site directed mutagenesis; tyrosine; Escherichia coli; Parkinson's disease; Pseudomonas; X ray crystallography; active sites; apoenzymes; binding sites; computer graphics /printing; covalent bond; electron transport; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; flavin adenine dinucleotide; flavoproteins; fluorimetry; heme; mitochondrial disease /disorder; oxidoreductase; oxygenases; protein purification; site directed mutagenesis; tyrosine

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to gain a thorough understanding of the physical, biochemical, kinetic and mechanistic properties of the bacterial flavocytochrome, p-cresol methylhydroxylase (PCMH). The primary goal will be to study important aspects of the assembly of PCMH, particularly those relating to the mechanism of covalent FAD attachment to Tyr384 in the flavoprotein subunit. Our second goal will be to study some aspects of substrate binding and the catalytic mechanism, particularly with the modified forms of PCMH and PchF made available for the primary goal. Adjunct studies of goals 1 & 2 will provide information concerning the mechanism of electron transfer from FAD to heme in PCMH. These investigations will involve pure site-specifically altered forms of the PCMH flavoprotein subunit, and pure forms of the wild-type subunit that contain altered flavin cofactors. These unnatural proteins will be studied by a variety of physical, chemical, and kinetic methods. Each of these proteins is designed to test the participation of a particular amino acid, or the property of the flavin in fundamentally important processes of PCMH. This research will be guided by the high-resolution x-ray crystal structures of fully and partially assembled forms of PCMH. PCMH provides a simple, convenient model for oxidoreductases, and specifically for human enzymes that contain covalently bound flavin as cofactors. These include the human mitochondrial enzymes succinate dehydrogenase, monoamine oxidase, dimethylgycine dehydrogenase and sarcosine dehydrogenase, and the human peroxisomal enzymes L-pipecolic acid oxidase and sarcosine oxidase. Information gathered from the studies of PCMH will provide insights into how the human enzymes assemble and function. This information may lead to the development of better treatments for some human afflictions, such as Parkinsonisn, depression, stress, mitochondrial myopathies, and aging.

描述（由申请人提供）：拟议的长期目标 研究是为了彻底了解物理，生化， 细菌黄素色素的动力学和机械特性，P-Cresol 甲基羟化酶（PCMH）。主要目标是研究重要方面 PCMH的组装，尤其是与 黄蛋蛋白亚基中的Tyr384的共价FAD附着。我们的第二个目标 将研究底物结合的某些方面和催化机制， 特别是使用PCMH和PCHF的修改形式可用于 主要目标。目标1和2的辅助研究将提供信息 关于电子从PCMH中从FAD到血红素转移的机理。 这些调查将涉及纯粹的现场特异性更改的形式 PCMH黄蛋蛋白亚基和包含的野生型亚基的纯形式 黄素辅因子改变。这些不自然的蛋白质将通过一种种类研究 物理，化学和动力学方法。这些蛋白质中的每一个都是设计的 测试特定氨基酸的参与或 黄素在PCMH的根本重要过程中。这项研究将是 以完全和部分的高分辨率X射线晶体结构为指导 PCMH组装形式。 PCMH为氧化还原酶提供了简单，方便的模型，特别是 对于包含共价结合黄素作为辅助因子的人类酶。这些 包括人线粒体酶琥珀酸脱氢酶，单胺 氧化酶，二甲基某二甲基脱氢酶和肌苷脱氢酶，以及 人过氧化物酶体酶L-Pipecolic氧化酶和肌苷氧化酶。 从PCMH的研究中收集的信息将提供有关如何 人类酶组装和功能。此信息可能会导致 为某些人类苦难开发更好的治疗方法，例如 帕金森尼，抑郁，压力，线粒体肌病和衰老。