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

基本信息

批准号：
8117523
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
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8117523
关键词：
Active Sites Adopted Amino Acids Anthrax disease Bacillus anthracis Bacteria Bacterial Spores Biochemical Process Biological Process Botulism Carbon Catalysis Chemistry Clostridium botulinum Coenzymes Coupled Crystallography DNA DNA Repair DNA Repair Enzymes DNA Repair Pathway DNA biosynthesis DNA photoproducts DNA-Protein Interaction Disease Endospore-Forming Bacteria Environment Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Excision Fermentation Food Poisoning Free Radicals Generations Germination Goals Human Hydrocarbons Isotopes Kinetics Measurable Mediating Methods Microbe Mutagenesis Names Nature Organic Chemistry Oxidation-Reduction Phase Proteins Reaction Reproduction spores Research Research Project Grants S-Adenosylmethionine Snoring Source Sterilization Structure Surveys Thymine Toluene analog base chemical kinetics crosslink enzyme mechanism in vivo inhibitor/antagonist medical schools novel pollutant prevent public health relevance repaired spore photoproduct lyase sugar tool ultraviolet damage ultraviolet irradiation

项目摘要

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.

Enzynnes利用有机自由基来催化各种重要的Nnetabolic反应。总体目标这项研究是为了描述这些酶的激进产生和控制的机械细节。这在ROO阶段进行的研究将集中于称为孢子光产物裂解酶（SPL）的DNA修复酶。 Spl 利用由唯一的[4FE-4S]簇结合的S-腺苷甲硫代（SAM）生成反应性有机物自由基修复独特的T-T交叉链接5-甲氨基-5，6-二甲氨氨氨酸（通常称为孢子光产物，在紫外线照射下形成的Sp）。 SPS存在于细菌的孢子中，例如枯草芽孢杆菌和炭疽芽孢杆菌。它采用了“直接反向”策略要修复SP，这意味着紫外线损坏很快就被拆卸也不替换受损的胸腺碱。因此，它代表了自然界中独特的DNA修复途径。另外，有效通过SPL催化的DNA修复使紫外线辐照不再致命孢子形成细菌。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，通过SP类似物探测反应机制（基于机理的酶抑制剂），检查动力学同位素效应和反应可逆性。此外，将确定[4FE-4S]群集的氧化还原电位和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确定潜在的SPL抑制剂。由于SPL是修复形成内孢子细菌紫外线损伤的关键酶，因此抑制其在体内活性将阻止细菌在发芽阶段固定这些损伤。与SPL抑制剂结合使用，紫外线辐射将其作为一种廉价且方便的灭菌工具，用于绝育。