Enzyme Catalysis of Toluene Degradation and Unusual DNA Photoproduct Repair

Enzynnes利用有机自由基催化各种重要的网络代谢反应。的总目标是 本研究旨在阐明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。SPL 利用S-腺苷蛋氨酸(SAM)与独特的[4Fe-4S]簇偶联生成反应性有机化合物 自由基修复T-T交联体特有的5-胸腺嘧啶-5，6-二氢胸腺嘧啶(俗称孢子 在紫外线照射下形成的光产物，SP)。 SPL存在于枯草杆菌和炭疽杆菌等细菌的孢子中。它采取了“直接反向”的战略。 以修复SP，这意味着紫外线损伤可以快速逆转，既不需要拆卸也不需要更换 胸腺嘧啶碱基受损。因此，它代表了自然界中一种独特的DNA修复途径。此外，高效的 SPL催化的DNA修复使紫外线对芽胞形成菌不再具有致死性。至 了解SPL介导的DNA修复反应、化学方法、动力学方法、光谱方法和诱变方法 将会被雇佣。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，用SP类似物(基于机制的酶)探索反应机理 抑制剂)，并检查动力学同位素效应和反应可逆性。此外，还将测定[4Fe-4S]团簇的氧化还原电势，以及SAIV！并对氧化还原电位的关键氨基酸进行了研究。 了解酶的作用机制将有助于我们发现潜在的SPL抑制剂。由于SPL是修复内孢子形成细菌紫外线损伤的关键酶，在体内抑制其活性将阻止细菌在萌发阶段修复这些损伤。与SPL抑制剂相结合，紫外线照射将重新成为一种廉价而方便的灭菌工具。

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.