Mechanisms of Radical SAM Enzymes Probed by EPR Spectroscopy

EPR 光谱探讨自由基 SAM 酶的作用机制

• 批准号: 8926453
• 负责人: R David Britt
• 金额: $ 30.79万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8926453
• 关键词: 9-mercaptodethiobiotin; Active Sites; Affect; Anabolism; Antibiotics; Anticodon; Arizona; Berlin; Binding; Biochemical; Biological; Biology; Biotin; Chemicals; Chemistry; Cleaved cell; Codon Nucleotides; Complex; Data Set; Divalent Cations; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electronics; Electrons; Environment; Enzymes; Equipment; Event; Excision; Family; Felis catus; Funding; Goals; Guanine; Hawaii; Health; Human; Hydrogen; Hydrogenase; Ions; Isotope Labeling; Kinetics; Label; Laboratories; Learning; Ligands; Light; Lyase; Magnetic Resonance; Magnetism; Mass Spectrum Analysis; Measures; Metals; Methods; Modeling; Modification; Nature; Nitrogen; Nucleoside Q; Oxygen; Pathway interactions; Play; Positioning Attribute; Preparation; Property; Protein Biosynthesis; Proteins; Protons; RNA; Radioisotopes; Reaction; Rest; S-Adenosylmethionine; Sampling; Sequence Analysis; Side; Site; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structure; Sulfur; Time; Transfer RNA; Tyrosine; United States National Institutes of Health; Universities; Vitamins; Work; base; biotin synthase; chemical reaction; design; desthiobiotin; divalent metal; electronic structure; enzyme activity; geometric structure; improved; instrument; interest; iron hydrogenase; research study; tetrahydropterin; thiophane; vitamin biosynthesis

DESCRIPTION (provided by applicant): Nature uses highly reactive radicals to carry out a diverse set of biochemical functions, many of which are essential to maintaining proper human health. These potent biological radical reactions need to be carried out safely, producing essential specific products, without dangerous side reactions occurring. A large number of such radical reactions are performed by the family of radical SAM enzymes, which use a [4Fe-4S] center with a bound S-adenosylmethionine (SAM) molecule to generate a strongly oxidizing 5'-deoxyadenosyl radical which can in turn drive a large number of difficult chemical reactions. We will target mechanistic aspects of several classes of radical SAM enzymes. Biotin synthase is a radical SAM enzyme that catalyzes the final step in the biosynthesis of the vitamin biotin. A set of Fe-S and radical SAM maturase enzymes are used to build the unique Fe-S center of Fe-Fe hydrogenase, an enzyme which catalyzes the important reduction of protons to dihydrogen and vice versa. And radical SAM enzymes are used to modify many bases in transfer RNA, improving codon-anticodon recognition in order to make protein synthesis more reliable. We are specifically interested in a radical SAM enzyme QueE that is essential for generating 7-deazapurines. This proposal describes a magnetic resonance spectroscopic approach to study such diverse radical SAM enzymes. Specifically, we are using electron paramagnetic resonance (EPR) spectroscopy, which can precisely measure the magnetic environment of unpaired electrons in the radical SAM Fe-S clusters, in the organic radicals that these clusters generate, and in secondary metal centers that are involved in the reactions in many of these enzymes.

描述（由申请人提供）：大自然使用高反应性自由基来执行多种生化功能，其中许多对于维持人类健康至关重要。这些有效的生物自由基反应需要安全地进行，产生重要的特定产物，而不发生危险的副反应。大量此类自由基反应是由自由基 SAM 酶家族进行的，该酶利用 [4Fe-4S] 中心与结合的 S-腺苷甲硫氨酸 (SAM) 分子生成强氧化性 5'-脱氧腺苷自由基，进而驱动大量困难的化学反应。我们将针对几类自由基 SAM 酶的机制方面进行研究。生物素合酶是一种自由基 SAM 酶，可催化维生素生物素生物合成的最后一步。一组 Fe-S 和自由基 SAM 成熟酶用于构建 Fe-Fe 氢化酶的独特 Fe-S 中心，该酶可催化质子重要还原为氢气，反之亦然。自由基SAM酶用于修饰转移RNA中的许多碱基，改善密码子-反密码子识别，从而使蛋白质合成更加可靠。我们对一种自由基 SAM 酶 QueE 特别感兴趣，它对于生成 7-脱氮嘌呤至关重要。该提案描述了一种磁共振波谱方法来研究此类不同的自由基 SAM 酶。具体来说，我们使用电子顺磁共振（EPR）光谱，它可以精确测量自由基 SAM Fe-S 团簇、这些团簇产生的有机自由基以及参与许多这些酶的反应。