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: 9132280
• 负责人: R David Britt
• 金额: $ 29.65万
• 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/9132280
• 关键词: 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; 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）：自然使用高反应性自由基来执行各种生化功能，其中许多功能对于维持适当的人类健康至关重要。这些有效的生物自由基反应需要安全地进行，生产基本的特定产品，而不会产生危险的侧面反应。自由基SAM酶的家族进行了大量的自由基反应，这些反应使用[4FE-4S]中心具有结合的S-腺苷甲硫代（SAM）分子来产生强烈的5'-脱氧腺苷自由基，从而产生大量困难化学反应。我们将针对几类自由基SAM酶的机械方面。生物素合酶是一种自由基SAM酶，它催化了维生素生物素的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，Fe-Fe氢化酶是一种酶，该酶催化了质子对二氢的重要还原，反之亦然。自由基SAM酶用于修饰转移RNA中的许多碱基，改善了密码子 - 抗浓度识别，以使蛋白质合成更可靠。我们特别对一种对产生7二氮嗪至关重要的自由基SAM酶Quee感兴趣。该提案描述了一种研究这种多种自由基SAM酶的磁共振光谱方法。具体而言，我们使用电子顺磁共振（EPR）光谱，可以精确地测量这些簇产生的有机自由基中不成对电子的磁性环境，以及在许多这些酶中涉及的二级金属中心中所产生的有机自由基。