De Novo Mini-Metalloenyzmes with Hydrolase Activity

具有水解酶活性的从头微型金属酶

• 批准号: 10359516
• 负责人: Katherine Michelle Buettner
• 金额: $ 38.21万
• 依托单位: GETTYSBURG COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359516
• 关键词: ATP Hydrolysis; Acidity; Active Sites; Affinity; Amino Acid Substitution; Amino Acids; Binding; Biochemical Reaction; Biological; Biological Assay; Biological Models; Biomimetics; Blood; Buffers; Catalysis; Cell physiology; Chemicals; Chemistry; DNA; Deoxyribonucleases; Development; Enzymes; Exposure to; Foundations; Future; Goals; Hydrogen Bonding; Hydrolase; Investigation; Ions; Journals; Libraries; Life; Metalloproteins; Metals; Modification; Molecular; Nature; Phosphoric Monoester Hydrolases; Process; Production; Protein Engineering; Proteins; Proteolysis; Publications; Reaction; Reporting; Role; Scaffolding Protein; Series; Solvents; Specificity; Structure-Activity Relationship; System; Techniques; Titanium; Training; Transition Elements; Vanadium; Water; Work; Zinc; aqueous; base; catalyst; design; experience; human disease; improved; insight; metalloenzyme; rare genetic disorder; reaction rate; scaffold; small molecule; symposium; trend; undergraduate student

PROJECT SUMMARY The major goal of the proposed work is to create, characterize, and optimize de novo binuclear mini- metalloenzymes for hydrolase activity. Hydrolytic cleavage, or the breaking apart of a molecule by the addition of water, is an essential biochemical reaction that governs cellular processes across all life kingdoms. Several rare genetic diseases come from hydrolase malfunctions, making them important to understand. Metal ions are frequently used to catalyze these reactions due to their ability to strongly polarize the O–H bond in water even at neutral pH. A small artificial enzyme called DFsc, which contains two metal ions in the active site, is capable of hydrolyzing small molecules and larger DNA substrates. Activity was observed with zinc, which is commonly used by nature, and titanium, which has no known natural function. This project seeks to (1) understand the structural and electronic factors that contribute to hydrolase activity in the DFsc system and (2) develop additional mini-metalloenzymes with metals underutilized by nature for environmentally-friendly and catalytically-efficient hydrolytic cleavage. To accomplish these goals, we will design, produce, and comprehensively characterize a series of DFsc proteins whose metal-coordinating amino acids are systematically varied. This library of proteins will be screened for hydrolase activity using DNAse and phosphatase assays and a variety of metal ions. Successful completion of these aims will result in a robust model system that will provide an understanding of the structure-activity relationships in natural and artificial metallohydrolases.

项目概要 拟议工作的主要目标是创建、表征和优化从头双核微型核 用于水解酶活性的金属酶。 加水是控制所有生命细胞过程的重要生化反应 一些罕见的遗传疾病是由水解酶功能障碍引起的，这使得它们对人类来说很重要。 金属离子因其强烈的催化能力而经常被用来催化这些反应。 即使在中性 pH 值下，也会使水中的 O-H 键极化，这种酶称为 DFsc。 活性位点含有两个金属离子，能够水解小分子和较大的DNA 观察到了自然界常用的锌和钛的活性。 没有已知的自然功能。该项目旨在（1）了解其结构和电子因素。 有助于 DFsc 系统中的水解酶活性，并且 (2) 开发额外的微型金属酶 自然界中未充分利用的金属，可用于环境友好且催化高效的水解裂解。 为了实现这些目标，我们将设计、生产并全面表征一系列 DFsc 蛋白质，其金属配位氨基酸系统地变化。 将使用 DNAse 和磷酸酶测定以及各种金属来筛选水解酶活性 成功完成这些目标将产生一个强大的模型系统，该系统将提供 了解天然和人工金属水解酶的结构-活性关系。