Mechanisms of Radical SAM Enzymes Probed by EPR Spectroscopy

项目摘要 自然界使用高活性自由基来执行各种生化功能，其中许多是埃森的， 有助于维持人体健康这些强有力的生物自由基反应需要安全地进行， 生产必需的特定产品，而不会发生危险的副反应。一大批这样的人-- 通过自由基SAM酶家族进行化学反应，其使用具有结合的[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， 转驱动大量困难的化学反应。我们将针对几类机械方面的 自由基SAM酶。生物素合成酶是一种自由基SAM酶，催化生物合成的最后一步 维生素生物素。一组Fe-S和自由基SAM成熟酶用于构建独特的Fe-S中心 铁-铁氢化酶是一种催化质子还原为二氢和二氢的重要酶， 亦然自由基SAM酶被用来修饰转移RNA中的许多碱基，提高密码子-反密码子的效率， 为了使蛋白质合成更可靠。我们对一种激进的SAM特别感兴趣 QueE酶是产生7-脱氮嘌呤所必需的。这个提议描述了一种磁共振 光谱方法来研究这种不同的自由基SAM酶。具体来说，我们使用电子帕拉- 磁共振（EPR）光谱，它可以精确地测量未配对的磁环境， 自由基SAM Fe-S簇中的电子，这些簇产生的有机自由基中的电子，以及次级自由基中的电子。 这些金属中心参与了许多酶的反应。

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.