Mechanisms of Iron-Containing Enzymes

项目总结/摘要 利用含铁辅因子的酶催化许多不同的、通常非常困难的化学反应， 对核心生命过程至关重要，如DNA生物合成和修复，基因调控， 表观遗传的调节，具有抗菌和抗真菌作用的多种化合物的生物合成 活动这些酶的功能障碍通常与严重疾病的发作有关，例如癌症， 心血管疾病和糖尿病。利用这些酶的合成潜力， 治疗与其功能障碍相关的疾病涉及合理操纵这些过程， 分子水平，这需要详细了解反应机制，特别是酶如何 控制他们反应的结果。Bollinger/Krebs联合小组专门研究将瞬态 快速动力学实验与各种光谱（如停流吸收，冷冻淬火EPR和 Mössbauer）和分析（LC/MS）方法来监测金属酶反应。在过去的20年里，我们的团队 已经成功地研究了许多单核（MNHI）和双核非血红素铁（DNHI）酶， 并表征其催化循环中的关键反应中间体。我们捕获并鉴定了高自旋 在各种MNHI酶中的Fe（IV）-氧代（铁酰基）中间体。铁基中间体引发底物氧化， 典型地通过脂肪族C-H键的断裂。这些反应的结果多种多样，包括 羟基化（默认结果）、卤化、去饱和、差向异构化、杂环化和 内过氧化反应我们目前的研究重点是破译导致 不同的结果。本研究的长期目标是为合理操作 这些酶用于生物技术应用。与Boal集团合作，B/K集团已 确定了血红素加氧酶二铁加氧酶（HDO）酶超家族，目前研究了几种 HDO。PI还与Booker小组就自由基S-的机制研究进行了长期合作。 腺苷甲硫氨酸（RS）酶，催化新的C-S键的形成。这些酶利用它们的[Fe 4S 4]RS 簇以产生典型的5 '-脱氧-腺苷-5'-基自由基，该自由基通过切割 脂肪族C-H键及其辅助[Fe_4S_4]簇合物作为硫源。当前合作的重点 对RS酶的研究工作旨在描述各种RS酶的反应机制， 形成新的C-S键。

Project Summary/Abstract Enzymes that utilize iron-containing cofactors catalyze many different, often very difficult, chemical reactions that are fundamentally important to central life processes, such as DNA biosynthesis and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of numerous compounds with antibacterial and antifungal activities. Dysfunction of these enzymes is often associated with the onset of severe diseases, e.g. cancer, cardiovascular diseases, and diabetes. Strategies to harness the synthetic potential of these enzymes and to treat diseases associated with their dysfunction involves the rational manipulation of these processes on a molecular level, which requires a detailed knowledge of the reaction mechanisms, in particular how the enzymes control the outcome of their reactions. The Bollinger/Krebs joint group specializes in combining transient-state rapid kinetic experiments with various spectroscopic (e.g. stopped-flow absorption, freeze-quench EPR and Mössbauer) and analytical (LC/MS) methods to monitor metalloenzyme reactions. In the last 20 years, our group has successfully studied many mononuclear (MNHI) and dinuclear non-heme-iron (DNHI) enzymes by trapping and characterizing key reaction intermediates in their catalytic cycles. We trapped and characterized high-spin Fe(IV)-oxo (ferryl) intermediates in various MNHI enzymes. The ferryl intermediate initiates substrate oxidation, typically by cleavage of an aliphatic C-H bond. The outcome of these reactions is diverse and includes hydroxylation (the default outcome), halogenation, desaturation, epimerization, heterocyclization, and endoperoxidation reactions. The current focus of our research aims at deciphering the factors that result in the diverse outcomes. The long-term goal of this research is to lay the foundation for the rational manipulation of these enzymes for biotechnological applications. In collaboration with the Boal group, the B/K group has identified the heme-oxygenase diiron oxygenase (HDO) enzyme superfamily and currently studies several HDOs. The PI also has a long-standing collaboration with the Booker group on mechanistic studies of radical S- adenosylmethionine (RS) enzymes that catalyze formation of new C-S bonds. These enzymes use their [Fe4S4]RS cluster to generate the canonical 5’-deoxy-adenos-5’-yl radical that initiates substrate oxidation by cleavage of aliphatic C-H bonds and their auxiliary [Fe4S4] clusters as sulfur source. The current focus of the collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of various RS enzymes involved in formation of new C-S bonds.