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多种重要的代谢反应。这项研究的总体目标是描绘这些酶的自由基生成和控制的机制细节。本研究主要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，其利用由独特的[4Fe-4S]簇偶联的S-腺苷甲硫氨酸（SAM）来产生反应性有机自由基。BSS存在于T. Aromatica通过蛋白质基甘氨酰自由基引发甲苯厌氧生物降解过程的机制将甲苯转化为苄基琥珀酸酯。由于甲苯及其相关化合物是具有神经元和致癌毒性的主要污染物类别，因此这项研究具有重要意义。为了产生甘氨酰自由基，需要活化酶（AE），其使用S ′-脱氧腺苷自由基氧化甘氨酸残基，所述S ′-脱氧腺苷自由基又通过用[4Fe-4S]簇提供的电子还原切割SAM而产生。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、光谱和致突变研究。具体目标包括酶动力学的研究，不同的[4Fe-4S]簇在BSS/BSS-AE中的作用，以及不同的有机自由基中间体在催化过程中的作用。SPL存在于细菌如B的孢子中。枯草芽孢杆菌，并修复独特的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）时形成的紫外线照射。SPL采用“直接反向”策略修复SP，既不涉及碱基或核苷酸的去除也不涉及替换。因此，它代表了自然界中独特的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。这些目标包括：研究SP在广泛的DNA二级结构中的修复，通过SP类似物（基于机制的酶抑制剂）探测反应机制，并检查动力学同位素效应和反应可逆性。此外，[4Fe-4S]团簇的氧化还原电位将被确定，SAM和关键氨基酸对氧化还原电位的影响将被研究。

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.