Mechanisms of Radical SAM Enzymes Probed by EPR Spectroscopy

描述(申请人提供)：大自然使用高活性的自由基来执行一系列不同的生化功能，其中许多是维持适当的人类健康所必需的。这些有效的生物自由基反应需要安全地进行，产生必要的特定产品，而不会发生危险的副反应。大量这样的自由基反应是由自由基自组胺酶家族执行的，它利用一个[4Fe-4S]中心与S-腺苷蛋氨酸(SAM)分子结合，产生一个强氧化性的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酶的机械方面。生物素合成酶是一种自由基SAM酶，催化维生素生物素生物合成的最后一步。利用一组Fe-S和自由基SAM成熟酶构建了Fe-Fe氢酶唯一的Fe-S中心，该酶催化重要的质子还原为二氢，反之亦然。而自由基SAM酶被用来修饰转移RNA中的许多碱基，提高密码子-反密码子的识别能力，从而使蛋白质合成更加可靠。我们特别感兴趣的是一种自由基SAM酶Quee，它是产生7-去氮嘌呤所必需的。这项建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。具体地说，我们正在使用电子顺磁共振光谱，它可以精确地测量自由基SAM Fe-S团簇中未配对电子的磁环境，这些团簇产生的有机自由基以及参与许多这些酶的反应的次级金属中心。

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.