Mechanisms of Mononuclear non-Heme-Iron Enzymes

项目摘要/摘要 利用含铁辅因子进行活性的酶催化了一系列令人眼花缭乱的(通常是非常 困难)对中心生命过程(如DNA生物合成)至关重要的化学反应 和修复，基因调节，表观遗传的调节，过量化合物的生物合成 抗菌和抗真菌活性)。这些酶的功能障碍通常与 严重疾病，如癌症、心血管疾病和糖尿病。驾驭合成纤维的策略 这些酶的潜力和对抗与其功能障碍相关的疾病涉及理性 在分子水平上操纵这些过程。这项工作的前提是要有详细的知识 了解潜在的反应机制，特别是酶如何控制其反应的结果。 Bollinger/Krebs联合小组专门将瞬变快速动力学实验与各种 光谱分析(如停流吸收、冷冻急冷EPR和穆斯堡尔谱)和分析(LC/MS) 方法对金属酶反应进行监测。在过去的15年里，他们的团队成功地研究了许多 需要单核或双核非血红素铁辅因子才能通过捕获和 表征催化循环中的关键反应中间体。特别是，他们发现了高自旋的Fe(IV)- 几种单核非血红素铁酶中的氧(铁)中间体，主要是铁(II)-和2-氧代-戊二酸- 依赖(Fe/2OG)酶。铁基中间体启动底物氧化，通常通过裂解一种 脂肪族C-H键。这些反应的结果是不同的，包括羟化(默认情况下 结果)、卤化、脱饱和、异构化和杂环化反应。这些反应中的许多 用于医学上重要的天然产物的生物合成。当前研究的重点是 布林格/克雷布斯小组的目标是破译导致不同结果的因素。的长期目标是 本研究为合理利用这些酶进行生物技术操作奠定了基础 申请。PI还与Squire Booker在Fe/S的机理研究上进行了长期的合作 酶，特别是属于自由基S-腺苷甲硫氨酸(RS)酶超家族的那些酶。 这些酶使用[4Fe-4S]簇来产生典型的5‘-脱氧腺苷-5’-基自由基，从而启动 多种多样的底物氧化，通常是通过裂解脂肪族的C-H键。《纽约时报》目前的重点 RS酶的合作研究旨在阐明不同反应的反应机理 结果，即硫插入、甲基化、甲硫化和去饱和化。

Project Summary/Abstract Enzymes that utilize iron-containing cofactors for their activity catalyze a bewildering array of (often very difficult) chemical reactions that are fundamentally important to central life processes (e.g., DNA biosynthesis and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of a plethora of 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 combat diseases associated with their dysfunction involves the rational manipulation of these processes on a molecular level. A prerequisite for this endeavor is a detailed knowledge of the underlying 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 15 years, their group has successfully studied many enzymes that require a mononuclear or a dinuclear non-heme-iron cofactor for activity by trapping and characterizing key reaction intermediates in their catalytic cycles. In particular, they identified high-spin Fe(IV)- oxo (ferryl) intermediates in several mononuclear non-heme-iron enzymes, mostly Fe(II)- and 2-oxo-glutarate- dependent (Fe/2OG) 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, and heterocyclization reactions. Many of these reactions are employed in the biosyntheses of medically important natural products. The current focus of research in the Bollinger/Krebs group 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. The PI also has a long-standing collaboration with Squire Booker on mechanistic studies of Fe/S enzymes, in particular those that belong to the superfamily of radical S-adenosylmethionine (RS) enzymes. These enzymes use a [4Fe-4S] cluster to generate a canonical 5’-deoxy-adenos-5’-yl radical that initiates a wide variety of substrate oxidations, often by cleavage of an aliphatic C-H bond. The current focus of the collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of different reaction outcomes, viz sulfur insertion, methylation, methylthiolation, and desaturation.