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: 8632910
• 负责人: R David Britt
• 金额: $ 31.59万
• 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/8632910
• 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; DNA Sequence Rearrangement; 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; public health relevance; research study; tetrahydropterin; thiophane; vitamin biosynthesis

Project Summary Nature uses highly reactive radicals to carry out a diverse set of biochemical functions, many of which are essen- tial 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 rad- ical 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 para- magnetic 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.

项目概要 大自然利用高反应性自由基来执行一系列不同的生化功能，其中许多功能是本质上的。 有助于维持适当的人类健康。这些有效的生物自由基反应需要安全地进行， 生产重要的特定产品，不会发生危险的副反应。大量此类辐射 化学反应是由自由基 SAM 酶家族进行的，该酶使用带有结合键的 [4Fe-4S] 中心。 S-腺苷甲硫氨酸 (SAM) 分子产生强氧化性 5'-脱氧腺苷自由基，可 进而驱动大量困难的化学反应。我们将针对几类的机械方面 自由基SAM酶。生物素合酶是一种自由基 SAM 酶，可催化生物合成的最后一步 维生素生物素。使用一组 Fe-S 和自由基 SAM 成熟酶来构建独特的 Fe-S 中心 Fe-Fe 氢化酶，一种催化质子重要还原为二氢和副氢的酶 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 识别以使蛋白质合成更加可靠。我们对激进的 SAM 特别感兴趣 QueE 酶对于生成 7-脱氮嘌呤至关重要。该提案描述了磁共振 光谱方法来研究此类不同的自由基 SAM 酶。具体来说，我们使用电子对位 磁共振（EPR）光谱，可以精确测量不成对的磁环境 自由基 SAM Fe-S 团簇中的电子、这些团簇产生的有机自由基中的电子以及次级团簇中的电子 参与许多这些酶的反应的金属中心。