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.

项目摘要 拟议工作的主要目的是创建，表征和优化从头binabler迷你 金属酶用于水解酶活性。水解裂解，或通过 加水，是一个基本的生化反应，它控制着所有生命的细胞过程 王国。几种罕见的遗传疾病来自水解酶故障，使其对 理解。金属离子经常用于催化这些反应，因为它们的能力很强 即使在中性pH下，水中的O – H键也会极化。一个称为DFSC的小型人造酶，它 在活性位点中包含两个金属离子，能够水解小分子和较大的DNA 基材。用锌观察到活性，锌通常被自然使用，钛有 没有已知的自然功能。该项目试图（1）了解结构和电子因素 有助于DFSC系统中的水解酶活性，（2）与其他微型 - 金甲酶一起使用 金属因自然而言是针对环保和催化有效的水解裂解的金属。 为了实现这些目标，我们将设计，制作和全面地描述一系列 DFSC蛋白的金属配位氨基酸系统变化。这个蛋白质库 将使用DNase和磷酸酶测定以及各种金属筛选水解酶活性 离子。这些目标的成功完成将导致强大的模型系统，该系统将提供 理解自然和人造金属水解酶中的结构活性关系。