Enzyme Catalysis of Toluene Degradation and Unusual DNA Photoproduct Repair

酶利用有机自由基催化多种重要的非代谢反应。的总目标 本研究旨在阐明这些酶的自由基产生和控制机制的细节。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。SPL 利用由独特的[4Fe-4S]簇偶联的S-腺苷甲硫氨酸（SAM）产生反应性有机 自由基来修复独特的T-T交联5-胸腺嘧啶-5，6-二氢胸腺嘧啶（通常称为孢子 光产物，SP）。 SPL存在于细菌如B的孢子中。枯草芽孢杆菌和B.炭疽病它采取的是“直接反向”策略 修复SP，这意味着紫外线的损害是迅速逆转，既不删除或更换的 受损的胸腺嘧啶碱基因此，它代表了自然界中独特的DNA修复途径。此外，高效 SPL催化的DNA修复使紫外线照射对产芽孢细菌不再致命。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。目的包括：调查SPL活动使用的基板具有广泛的 DNA二级结构，通过SP类似物探测反应机理（基于机理的酶 抑制剂），并检查动力学同位素效应和反应可逆性。此外，氧化还原电位的[4Fe-4S]集群将被确定和SAIV！和关键氨基酸的氧化还原电位将进行研究。 了解酶的机制将有助于我们识别潜在的SPL抑制剂。由于SPL是内孢子形成细菌中修复紫外线损伤的关键酶，因此在体内抑制其活性将防止细菌在萌发阶段修复这些损伤。结合SPL抑制剂，紫外线照射将恢复其作为一种廉价和方便的工具用于灭菌目的的权力。

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.