Study of copper oxidoreductases through metalloprotein engineering

通过金属蛋白工程研究铜氧化还原酶

• 批准号: 8231062
• 负责人: Steven M Berry
• 金额: $ 31.12万
• 依托单位: UNIVERSITY OF MINNESOTA DULUTH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231062
• 关键词: Active Sites; Address; Azurin; Binding; Biochemical; Biological Assay; Biotechnology; Budgets; Ceruloplasmin; Characteristics; Chemistry; Copper; Disease; Dopamine; Electron Transport; Elements; Engineering; Enzymes; Exhibits; Family; Goals; Human; Human body; Indium; Lead; Ligands; Light; Mediating; Mentors; Metalloproteins; Metals; Methodology; Mixed Function Oxygenases; Modeling; Modification; Molecular; Monophenol Monooxygenase; Mutagenesis; Mutation; Nitrite Reductase; Oxidation-Reduction; Oxidoreductase; Oxygen; Pathway interactions; Process; Property; Protein Family; Proteins; Reaction; Recording of previous events; Research; Roentgen Rays; Role; Scaffolding Protein; Science; Scientist; Site; Structure; Structure-Activity Relationship; Students; System; Testing; Trace Elements; Training; Work; amine oxidase; biological systems; career; cytochrome c oxidase; design; enzyme mechanism; enzyme model; experience; meetings; metalloenzyme; multidisciplinary; next generation; oxidation; peptidylglycine alpha-amidating monooxygenase; scaffold; tool

DESCRIPTION (provided by applicant): We will use metalloprotein design and redesign to address important functional features common to many copper metalloenzymes. Copper proteins are an important class of metallobiomolecules with at least a dozen enzymes in the human body utilizing copper for structure and function. Most human copper enzymes are classified as oxidoreductases and are involved in an array of processes, such as electron transfer, and oxidation or reduction of substrates. Common roles of copper in these proteins are electron transfer, such as in ceruloplasmin (CP) or cytochrome c oxidase (CcO), and reactivity with O2, such as in peptidyl ?-hydroxylating monooxygenase (PHM), tyrosinase, or amine oxidases. Therefore, themes common among Cu enzymes include 1) Cu mediated electron transfer and 2) Cu catalyzed oxygen chemistry with both oxidation and reduction of substrate. The goal of this proposal is to use protein design and redesign to explore and elucidate these common themes of Cu metalloenzymes. We will accomplish this by modeling the family of noncoupled dinuclear copper proteins, which contain both an electron transfer center and a catalytic center. We therefore will model the di-copper centers of PHM, dopamine ?-monooxygenase (DBM), and nitrite reductase (NiR) using the protein azurin as the ligand or scaffold. Using the protein design methodology, we will model these enzymes structurally as well as functionally. We will examine and adjust different layers of structural elements in our models that are hypothesized to be essential for activity in the native systems. These include 1) modulating the copper redox potentials of the two Cu sites, 2) tuning the electron transfer pathway between the Cu sites, and 3) making mutations to encourage substrate binding in our models. All of these components taken together are attempts to couple together and test different factors that are deemed important for the proper function of a catalytic Cu enzyme: the design of a structurally adequate copper site, the tuning of redox potential, the modification of electron transfer paths, and the binding of substrates such as oxygen. The long term goal of the research is to understand these properties of Cu metalloenzymes and shed light on the mechanisms of the native systems. Equally as important a goal for these projects is the mentoring and training of undergraduate and M.S. students for successful careers in multidisciplinary biochemical sciences. PUBLIC HEALTH RELEVANCE: There are over a dozen human copper enzymes, many of which utilize copper to facilitate electron transfer reactions and oxidize substrate with molecular oxygen. We will use protein metal site design to incorporate and adjust different layers of structural elements into models of native systems that test factors hypothesized to be essential for function. Defining the features that lead to activity of copper in these enzymes can contribute to an understanding of factors underlying various diseases.

描述(由申请人提供)：我们将使用金属蛋白设计和重新设计，以解决许多铜金属酶共同的重要功能特征。铜蛋白是一类重要的金属生物分子，人体内至少有十几种酶利用铜的结构和功能。大多数人类铜酶被归类为氧化还原酶，参与一系列过程，如电子转移和底物的氧化或还原。铜在这些蛋白质中的常见作用是电子传递，如铜蓝蛋白(CP)或细胞色素C氧化酶(CcO)，以及与O2的反应，如肽羟化单加氧酶(PHM)、酪氨酸酶或胺氧化酶。因此，铜酶的共同主题包括：1)铜介导的电子传递；2)铜催化底物氧化和还原的氧化学。这项提议的目的是利用蛋白质设计和重新设计来探索和阐明铜金属酶的这些共同主题。我们将通过对非偶联双核铜蛋白家族进行建模来实现这一点，该家族包含一个电子转移中心和一个催化中心。因此，我们将使用天青蛋白作为配体或支架来模拟PHM、多巴胺单加氧酶(DBM)和亚硝酸盐还原酶(NIR)的双铜中心。使用蛋白质设计方法，我们将从结构和功能上对这些酶进行建模。我们将检查和调整我们的模型中假定对本机系统中的活动至关重要的不同层的结构元素。这些措施包括1)调节两个铜位的铜氧化还原电位，2)调节铜位之间的电子转移路径，以及3)在我们的模型中进行突变以鼓励底物结合。所有这些成分都试图结合在一起，并测试不同的因素，这些因素被认为是催化铜酶正常功能的重要因素：结构适当的铜位置的设计，氧化还原电位的调节，电子转移路径的修改，以及底物(如氧)的结合。这项研究的长期目标是了解铜金属酶的这些性质，并阐明自然系统的机制。同样重要的是，这些项目的一个目标是指导和培训本科生和硕士研究生，使他们在多学科的生化科学领域取得成功。 与公共健康相关：人类有十几种铜酶，其中许多利用铜来促进电子转移反应，并用分子氧氧化底物。我们将使用蛋白质金属位点设计，将不同层次的结构元素合并并调整到本地系统的模型中，以测试假设对功能至关重要的因素。确定导致这些酶中铜的活性的特征有助于理解各种疾病的潜在因素。