Mechanisms of Mononuclear non-Heme-Iron Enzymes

单核非血红素铁酶的机制

• 批准号: 10394263
• 负责人: CARSTEN KREBS
• 金额: $ 49.25万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394263
• 关键词: Anabolism; Anti-Bacterial Agents; Antibiotics; Antifungal Agents; Biotechnology; Cardiovascular Diseases; Chemicals; Collaborations; DNA Repair Gene; DNA biosynthesis; Diabetes Mellitus; Disease; Enzymes; Foundations; Freezing; Functional disorder; Gene Expression Regulation; Goals; Health; Human; Hydrogen Bonding; Hydroxylation; Iron; Joints; Kinetics; Knowledge; Life; Malignant Neoplasms; Medical; Methods; Methylation; Molecular; Monitor; Mononuclear; Natural Products; Outcome; Oxygen; Process; Reaction; Regulation; Research; S-Adenosylmethionine; Societies; Sulfur; absorption; alpha ketoglutarate; chemical reaction; cofactor; combat; epigenetic regulation; epimerization; experimental study; halogenation; liquid chromatography mass spectrometry; metalloenzyme; oxidation

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.

项目摘要/摘要 利用含铁辅助因子的活性的酶会催化迷人的阵列（通常非常非常 难度）化学反应对中央生命过程至关重要（例如，DNA生物合成 和修复，基因调节，表观遗传遗传的调节，多种化合物的生物合成 抗菌和抗真菌活性）。这些酶的功能障碍通常与 严重疾病，例如癌症，心血管疾病和糖尿病。利用合成的策略 这些酶的潜力并打击与它们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。这项工作的先决条件是详细的知识 基础反应机制，特别是酶如何控制其反应结果。 Bollinger/Krebs联合组专门将瞬态快速动力学实验与各种相结合 光谱学（例如停止流量抽象，冻结EPR和Mössbauer）和分析（LC/MS） 监测金属酶反应的方法。在过去的15年中，他们的小组成功研究了许多 需要捕获和 表征关键反应中间的催化周期中。特别是，他们确定了高自旋Fe（IV） - Oxo（Ferryl）中间体中的几种单核非血红素铁酶，主要是Fe（II）和2-氧气 - 谷物 依赖（Fe/2og）酶。渡轮中间体会引发底物氧化，通常是通过裂解 脂肪族C-H键。这些反应的结果是潜水员，包括羟基化（默认 结果），卤素化，宗教，发作和杂环反应。其中许多反应 用于医学重要天然产品的生物合成。当前研究重点 Bollinger/Krebs小组的目标是破译导致潜水员结果的因素。长期目标 这项研究是为这些酶合理操纵生物技术的基础 申请。 PI还与Squire Booker进行了长期的合作，涉及Fe/S的机理研究 酶，尤其是属于自由基S-腺苷硫氨酸（RS）酶超家族的酶。 这些酶使用[4FE-4S]群集生成启动A的规范5'-脱氧-Adenos-5'-yl-ill激素 通常通过脂肪族C-H键的裂解，多种底物氧化。当前的重点 RS酶的合作研究工作旨在描述不同反应的反应机制 结果，硫插入，甲基化，甲基化和去饱和度。