Incorporation of Selenium into Xanthine Dehydrogenase

虽然最近在硒蛋白中结合硒的研究取得了很大进展，但这项工作的重点主要放在以硒半胱氨酸形式携带硒的酶上。少数几种酶的特征是，硒存在并需要活性，但在mRNA翻译过程中不插入，因此不以硒半胱氨酸的形式存在。两种这样的酶是来自梭状芽孢杆菌的烟酸羟基酶(NAH)和黄嘌呤脱氢酶(XD)。NaH中的硒在某种程度上是不稳定的，可以通过还原剂，如二硫苏糖醇，或通过处理杂色剂，如十二烷基硫酸钠来去除。为了更好地了解这种结合硒的性质，我们打算从紫色梭菌中分离和鉴定黄嘌呤脱氢酶(XD)，并利用该酶来鉴定将硒结合到辅酶中所需的成分。已知的是，紫色梭菌具有结构性的XD活性，当在生长介质中添加亚硒酸盐时，这种活性会增加。一旦被分离，该酶将从亚基组成、辅因子分析(钼、铁-S簇，FAD)、底物专一性、酶动力学分析以及最重要的是鉴定存在于活性酶中的硒部分方面进行表征。从以亚硒酸盐形式存在的放射性标记的75-Se存在下生长的细胞中提纯酶将允许有效地分析硒的形式。一旦建立了从酶制剂中有效去除硒的条件，很可能是通过还原剂(S)处理，酶促XD就可以用作一种测定方法，以确定掺入硒所需的必要成分。这项工作可能会发现XD和其他钼羟基酶所需的一种新的递送蛋白(S)或硒供体分子，并可能揭示硒在硒半胱氨酸生物合成中的递送。此外，在从产甲烷万尼氏甲烷球菌中分离磷酸硒合成酶的过程中发现了一种新的蛋白质，该蛋白质也含有不稳定的硒。测定了该蛋白的N-末端氨基酸序列，与Genbank数据库中的任何一种蛋白几乎没有相似性。我们推测，该蛋白可能代表一种类似于XD的酶的亚基，在XD中，硒是不稳定的，不以硒半胱氨酸的形式存在。另一种可能性是，该蛋白是一种硒结合/递送蛋白，在硒半胱氨酸的生物合成过程中或在将硒掺入非硒半胱氨酸硒蛋白(如XD)的过程中，它与硒化合物结合。对该蛋白质的进一步分离和研究将揭示有关细胞内硒加工的新信息。-硒、黄嘌呤脱氢酶、梭状芽孢杆菌、钼羟基酶

Although significant knowledge has been gained recently on the incorporation of selenium into selenoproteins, most of the emphasis of this work has been placed on enzymes that carry selenium in the form of selenocysteine. A handful of enzymes have been characterized in which selenium is present and required for activity but is not inserted during translation of the mRNA and therefore is not present as selenocysteine. Two such enzymes are nicotinic acid hydroxylase (NAH) and xanthine dehydrogenase (XD) from Clostridium. Selenium present in NAH is somewhat labile and can be removed by reducing agents, such as dithiothreitol, or by treatment with chaotropic agents, such as sodium dodecyl sulfate. In order to better understand the nature of this incorporation of selenium, we intend to isolate and characterize xanthine dehydrogenase (XD) from Clostridium purinolyticum and use this enzyme to help characterize the components necessary to incorporate selenium into the apoenzyme. Clostridium purinolyticum is known to possess a constitutive XD activity that increases when selenite is supplemented to the growth medium. Once isolated, this enzyme will be characterized with respect to subunit composition, cofactor analysis (molybdenum, Fe-S clusters, FAD), substrate specificity, enzyme kinetic analysis, and, most importantly, identification of the selenium moiety present in the active enzyme. Purifying the enzyme from cells grown in the presence of radiolabeled 75-Se in the form of selenite will allow efficient analysis of the form of selenium. Once conditions can be established for efficient removal of the selenium from the enzymatic preparation, most likely by treatment with reducing agent(s), the apoenzyme XD can then be used as a type of assay to determine the necessary components required for incorporation of selenium. This work may uncover a new delivery protein(s) or selenium donor molecule required for XD and other molybdenum hydroxylases and perhaps shed light on the delivery of selenium in the biosynthesis of selenocysteine. In addition, a novel protein, which also contained labile selenium, was uncovered during isolation of selenophosphate synthetase from the methanogen Methanococcus vannielii. The N-terminal amino acid sequence of this protein was determined and had little similarity to any protein in the Genbank database. We speculate that this protein may represent a subunit of an enzyme similar to XD in which selenium is labile and not present as selenocysteine. Another possibility exists that this protein is a selenium binding/delivery protein that binds to a selenium compound during the process of biosynthesis of selenocysteine or in the incorporation of selenium into non-selenocysteine selenoproteins, such as XD. Further isolation and study of this protein should reveal new information on the processing of selenium within the cell. - selenium, xanthine dehydrogenase, Clostridium, molybdenum hydroxylase