Iron-Sulfur Clusters in Biological Radical Generation

生物自由基生成中的铁硫簇

• 批准号: 7931051
• 负责人: Joan B Broderick
• 金额: $ 33.56万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931051
• 关键词: Anabolism; Bacteria; Binding; Biochemical; Biological; Biology; Catalysis; Cells; Chemicals; Chemistry; Cleaved cell; Complex; Data; Data Reporting; Electron Nuclear Double Resonance; Electronics; Enzymes; Freezing; Funding; Generations; Human; Investigation; Iron; Label; Nature; Phylogenetic Analysis; Physiological; Property; Reaction; Roentgen Rays; Role; S-Adenosylmethionine; Sampling; Site; Spectrum Analysis; Sulfur; Techniques; Thioctic Acid; X-Ray Crystallography; absorption; analog; formate acetyltransferase activating enzyme; heme biosynthesis; molybdenum cofactor; mutant; novel; oxidation; peptide analog

DESCRIPTION (provided by applicant): The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (the so-called radical SAM) superfamily of enzymes. These enzymes span a remarkably diverse range of reactions and appear to be represented across the phylogenetic kingdom, with hundreds of radical SAM enzymes identified. The widespread occurrence of these enzymes throughout biology, from bacteria to humans, is indicative of the significance of the chemistry catalyzed by these enzymes. In humans, radical SAM enzymes are involved in the biosynthesis of lipoic acid, the synthesis of heme, and the biosynthesis of the molybdopterin cofactor, among many other essential functions, some as yet unidentified. Despite the diversity of reactions catalyzed, our overriding hypothesis is that the adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with S-adenosylmethionine to generate an adenosyl radical intermediate, which is directly involved in catalysis. These reactions represent novel chemistry for iron-sulfur clusters. To investigate this novel chemistry, biochemical, spectroscopic, mechanistic, and structural studies of pyruvate formate-lyase activating enzyme (PFL-AE) will be pursued. The specific aims include further investigation of the [4Fe-4S] cluster of PFL-AE, its unique iron site, its interactions with substrate, and its role in catalysis. In addition we propose to explore the nature of the PFL-AE iron-sulfur cluster in whole cells. We will also utilize substrate analogs as probes of ES interactions and the PFL-AE catalytic mechanism. We will use spectroscopic approaches, particularly ENDOR, to probe the structural and electronic features of the PFL- AE/AdoMet/PFL complex. We will also utilize cryoreduction and rapid-freeze-quench spectroscopic approaches to identify and spectroscopically characterize intermediates in the radical-generation reaction catalyzed by PFL-AE. Finally, we will pursue structural characterization of PFL-AE and its complexes with PFL and SAM using X-ray crystallography.

描述（由申请人提供）：本项目的总体目标是描述Fe/S-S-腺苷甲硫氨酸（所谓的自由基SAM）超家族酶产生自由基的详细化学机制。这些酶跨越一个显着不同的反应范围，并出现在整个系统发育王国的代表，与数百个激进的SAM酶确定。从细菌到人类，这些酶在整个生物学中的广泛存在表明了这些酶催化的化学作用的重要性。在人类中，自由基SAM酶参与了硫辛酸的生物合成、血红素的合成和异蝶呤辅因子的生物合成，以及许多其他基本功能，其中一些尚未鉴定。尽管催化的反应的多样性，我们压倒一切的假设是，腺苷甲硫氨酸依赖性铁硫酶都通过一个共同的机制，其中减少集群与S-腺苷甲硫氨酸相互作用，产生腺苷自由基中间体，这是直接参与催化。这些反应代表了铁硫簇合物的新化学。为了研究这种新的化学，生化，光谱，机制，和结构的丙酮酸甲酸裂解酶激活酶（PFL-AE）的研究将继续进行。具体目标包括进一步研究PFL-AE的[4Fe-4S]簇，其独特的铁位点，其与底物的相互作用，以及其在催化中的作用。此外，我们建议探索PFL-AE铁硫簇在整个细胞的性质。我们还将利用底物类似物作为ES相互作用和PFL-AE催化机制的探针。我们将使用光谱学方法，特别是ENDOR，来探测PFL-AE/C3Met/PFL复合物的结构和电子特征。我们还将利用低温还原和快速冷冻淬火光谱方法来识别和光谱表征PFL-AE催化的自由基生成反应中的中间体。最后，我们将使用X射线晶体学来研究PFL-AE及其与PFL和SAM的复合物的结构表征。