Enzyme Catalysis of Toluene Degradation and Unusual DNA Photoproduct Repair

描述（由申请人提供）：酶利用有机自由基催化各种重要的代谢反应。本研究的总体目标是描述这些酶产生和控制自由基的机制细节。本研究重点研究了三种自由基酶：K99期的苄基琥珀酸合成酶（BSS）及其激活酶（AE）和ROD期的孢子光产物裂解酶（SPL）。后两种酶属于自由基SAM超家族，它们利用s -腺苷蛋氨酸（SAM）与独特的[4Fe-4S]簇偶联产生活性有机自由基。在T. Aromatica等细菌中发现的BSS，通过一种涉及基于蛋白质的甘酰基自由基的机制，将甲苯转化为琥珀酸苄酯，从而启动甲苯的厌氧生物降解过程。这项研究具有重要意义，因为甲苯及其相关化合物是一类具有神经毒性和促癌毒性的主要污染物。为了生成甘氨酸自由基，需要一种激活酶（AE），它利用SAM的还原裂解产生的S'-脱氧腺苷自由基氧化甘氨酸残基，并利用[4Fe-4S]簇提供的电子。为了研究BSS催化的甲苯生物修复过程，将对BSS和BSS- ae进行生化、光谱和诱变研究。具体目的包括酶动力学的研究，不同[4Fe-4S]簇在BSS/BSS- ae中的功能，以及不同有机自由基中间体在催化过程中的作用。SPL存在于枯草芽孢杆菌等细菌的孢子中，修复紫外线照射下形成的独特的T-T交联的5-胸腺嘧啶- 5,6 -二氢胸腺嘧啶（通常称为孢子光产物，SP）。SPL采用“直接反向”策略来修复SP，既不涉及移除也不涉及替换碱基或核苷酸。因此，它代表了自然界中一种独特的DNA修复途径。为了了解SPL介导的DNA修复反应，将采用化学、动力学、光谱和诱变方法。研究目标包括：研究广泛的DNA二级结构中的SP修复，通过SP类似物（基于机制的酶抑制剂）探索反应机制，并研究动力学同位素效应和反应可逆性。此外，还将测定[4Fe-4S]簇的氧化还原电位，并研究SAM和关键氨基酸对氧化还原电位的影响。

DESCRIPTION (provided by applicant): Enzymes utilize organic radicals to catalyze a variety of important metabolic reactions. The overall goal of this research is to delineate the mechanistic details of radical generation and control by these enzymes. This research focuses on three radical enzymes: benzylsuccinate synthase (BSS) and its activating enzyme (AE) in the K99 phase, and spore photoproduct lyase (SPL) in the ROD phase. The later two enzymes belong to the radical SAM superfamily, which utilize S-adenosylmethionine (SAM) coupled by a unique [4Fe-4S] cluster to generate the reactive organic radicals. BSS, found in bacteria like T. Aromatica, converts toluene to benzylsuccinate by a mechanism involving protein-based glycyl radical to initiate the toluene anaerobic biodegradation process. This research is of significance due to the fact that toluene and its related compounds comprise a major category of pollutants with neuronal and cancer-promoting toxicity. To generate the glycyl radical, an activating enzyme (AE) is needed, which oxidizes the glycine residue using a S'-deoxyadenosyl radical generated, in turn by reductive cleavage of SAM with the electron provided by a [4Fe-4S] cluster. To investigate this BSS-catalyzed toluene bioremediation process, biochemical, spectroscopic, and mutagenic studies of both BSS and BSS-AE will be pursued. The specific aims include the investigations of enzyme kinetics, the functions of different [4Fe-4S] clusters in BSS/BSS-AE, and the roles of different organic radical intermediates during the catalysis. SPL exists in the spores of bacteria such as B. subtilis, and repairs unique T-T crosslink 5-thyminyl-5, 6- dihydrothymine (commonly called spore photoproduct, SP) formed upon UV irradiation. SPL adopts a "direct reverse" strategy to repair SP, which involves neither removal nor replacement of bases or nucleotides. It thus represents a unique DNA repair pathway in Nature. To understand the SPL mediated DNA repair reaction, chemical, kinetic, spectroscopic, and mutagenic methods will be employed. The objectives include: investigating the SP repair in 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 SAM and key amino acids to the redox potential will be investigated.