Enzyme Catalysis of Toluene Degradation and Unusual DNA Photoproduct Repair

酶催化甲苯降解和异常 DNA 光产物修复

• 批准号: 7881168
• 负责人: Lei Li
• 金额: $ 24.9万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7881168
• 关键词: Activase; Adopted; Amino Acids; Anthrax disease; Bacillus anthracis; Bacteria; Botulism; Catalysis; Clostridium botulinum; Coupled; DNA; DNA Repair; DNA Repair Enzymes; DNA Repair Pathway; DNA photoproducts; Disease; Endospore-Forming Bacteria; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Excision; Food Poisoning; Generations; Germination; Goals; Grant; Human; Isotopes; Kinetics; Label; Mediating; Methods; Names; Nature; Oxidation-Reduction; Peptides; Phase; Process; Reaction; Recombinants; Reproduction spores; Research; Snoring; Sterilization for infection control; Structure; Thymine; Toluene; analog; base; chemical kinetics; crosslink; enzyme mechanism; in vivo; inhibitor/antagonist; prevent; public health relevance; repaired; spore photoproduct lyase; tool; ultraviolet damage; ultraviolet irradiation

Enzynnes utilize organic radicals to catalyze a variety of important nnetabolic reactions. The overall goal of this research is to delineate the mechanistic details of radical generation and control by these enzymes. This research in the ROO phase will focus on a DNA repair enzyme named spore photoproduct lyase (SPL). SPL utilizes S-adenosylmethionine (SAM) coupled by a unique [4Fe-4S] cluster to generate the reactive organic radicals to repair the unique T-T crosslink 5-thyminyl-5, 6-dihydrothymine (commonly called spore photoproduct, SP) formed upon UV irradiation. SPL exists in the spores of bacteria such as B. subtilis and B. anthracis. It adopts a "direct reverse" strategy to repair SP, meaning that the UV damage is quickly reversed with neither removal nor replacement of the damaged thymine bases. It thus represents a unique DNA repair pathway in Nature. In addition, the efficient DNA repair catalyzed by SPL makes UV irradiation no longer lethal to the spore-forming bacteria. To understand the SPL mediated DNA repair reaction, chemical, kinetic, spectroscopic, and mutagenic methods will be employed. The objectives include: investigating the SPL activity using substrates with a wide range of DNA secondary structures, probing the reaction mechanism by SP analogues (mechanism-based enzyme inhibitors), and examining the kinetic isotope effects and reaction reversibility. In addition, the redox potential of the [4Fe-4S] cluster will be determined and the influence of SAIV! and key amino acids to the redox potential will be investigated. Understanding the enzyme mechanism will help us identify potential SPL inhibitors. As SPL is the key enzyme to repair the UV damage in endospore-forming bacteria, inhibiting its activity in vivo will prevent the bacteria from fixing these damages at the germination phase. In combination with the SPL inhibitor, UV irradiation will regain its power as a cheap and convenient tool for sterilization purpose.

酶利用有机自由基来催化各种重要的代谢反应。总体目标为 这项研究旨在描述这些酶产生和控制自由基的机制细节。这 ROO阶段的研究将集中在一种名为孢子光产物裂解酶（SPL）的DNA修复酶上。声压级 利用独特的 [4Fe-4S] 簇偶联的 S-腺苷甲硫氨酸 (SAM) 生成反应性有机化合物 自由基修复独特的 T-T 交联 5-胸腺嘧啶基-5, 6-二氢胸腺嘧啶（通常称为孢子 光产物，SP）在紫外线照射下形成。 SPL存在于枯草芽孢杆菌和炭疽芽孢杆菌等细菌的孢子中。它采用“直接反向”策略 修复 SP，这意味着无需移除或更换即可快速逆转紫外线损伤 胸腺嘧啶碱基受损。因此，它代表了自然界中独特的 DNA 修复途径。此外，高效的 SPL 催化的 DNA 修复使得紫外线照射不再对孢子形成细菌具有致命性。到 了解 SPL 介导的 DNA 修复反应、化学、动力学、光谱和诱变方法 将被雇用。目标包括： 使用具有广泛范围的底物研究 SPL 活性 DNA二级结构，利用SP类似物（机制酶）探究反应机制 抑制剂），并检查动力学同位素效应和反应可逆性。此外，将测定[4Fe-4S]团簇的氧化还原电位以及SAIV的影响！将研究氧化还原电位的关键氨基酸。 了解酶机制将有助于我们识别潜在的 SPL 抑制剂。由于SPL是内生孢子形成细菌修复紫外线损伤的关键酶，抑制其体内活性将阻止细菌在萌发阶段修复这些损伤。与 SPL 抑制剂结合使用，紫外线照射将重新发挥其作为一种廉价且方便的灭菌工具的作用。