Enzyme Catalysis of Toluene Degradation and Unusual DNA Photoproduct Repair

描述(申请人提供)：酶利用有机自由基催化各种重要的代谢反应。这项研究的总体目标是描绘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相的苄基琥珀酸合成酶(BSS)及其活化酶(AE)和棒状相的孢子光产物裂解酶(SPL)。后两种酶属于自由基SAM超家族，利用S-腺苷甲硫氨酸(SAM)与独特的[4Fe-4S]簇偶联生成反应性有机自由基。在T.aromatica等细菌中发现的BSS，通过一种涉及蛋白质基甘氨基自由基的机制将甲苯转化为丁二酸苄酯，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构成了一种主要的污染物类别，具有神经毒性和致癌毒性。为了产生甘氨酰自由基，需要一个活化酶(AE)，它使用S脱氧腺苷自由基氧化甘氨酸残基，该自由基是通过SAM与[4Fe-4S]簇提供的电子还原裂解而产生的。为了研究这种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。具体目标包括酶动力学的研究，不同[4Fe-4S]团簇在BSS/BSS-AE中的作用，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交联物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”策略修复SP，既不涉及碱基或核苷酸的移除或替换。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、光谱和诱变等方法。目的包括：研究广泛DNA二级结构中的SP修复，探讨SP类似物(基于机理的酶抑制剂)的反应机理，以及检测动力学同位素效应和反应可逆性。此外，还将测定[4Fe-4S]团簇的氧化还原电位，并考察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.