Incorporation of Selenium into Xanthine Dehydrogenase

将硒掺入黄嘌呤脱氢酶

• 批准号: 6227992
• 负责人: William T Self
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227992
• 关键词: Clostridium; active sites; apoenzymes; cofactor; enzyme activity; enzyme biosynthesis; enzyme substrate; flavin adenine dinucleotide; iron sulfur protein; metal metabolism; microorganism culture; molybdenum; protein biosynthesis; protein structure function; radionuclides; reducing agents; selenium; selenoprotein; xanthine dehydrogenase

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

虽然最近已经获得了重要的知识硒纳入硒蛋白，这项工作的重点是放在酶，携带硒的硒半胱氨酸的形式。少数酶的特征在于其中存在硒并且是活性所需的，但在mRNA的翻译期间不插入，因此不作为硒代半胱氨酸存在。两种这样的酶是来自梭菌属的烟酸羟化酶（NAH）和黄嘌呤脱氢酶（XD）。存在于NAH中的硒是稍微不稳定的，并且可以通过还原剂（例如二硫苏糖醇）或通过用离液剂（例如十二烷基硫酸钠）处理来去除。为了更好地了解硒的这种结合的性质，我们打算分离和表征黄嘌呤脱氢酶（XD）从梭菌嘌呤解，并使用这种酶，以帮助表征必要的组成部分，将硒纳入脱辅基酶。已知溶嘌呤梭菌具有组成型XD活性，当向生长培养基中补充亚硒酸盐时，该活性增加。一旦分离，该酶将通过亚基组成、辅因子分析（钼、Fe-S簇、FAD）、底物特异性、酶动力学分析以及最重要的是鉴定活性酶中存在的硒部分来表征。从在亚硒酸盐形式的放射性标记的75-Se的存在下生长的细胞中纯化酶将允许硒的形式的有效分析。一旦可以建立从酶制剂中有效去除硒的条件，最可能的是通过用还原剂处理，脱辅基酶XD然后可以用作一种类型的测定，以确定掺入硒所需的必要组分。这项工作可能会发现XD和其他钼羟化酶所需的新递送蛋白或硒供体分子，并可能揭示硒半胱氨酸生物合成中硒的递送。此外，一种新的蛋白质，其中也含有不稳定的硒，被发现在分离的硒磷酸合成酶产甲烷菌甲烷球菌vannielii。该蛋白的N-末端氨基酸序列与Genbank数据库中的任何蛋白都没有相似性。我们推测，这种蛋白质可能代表一种类似于XD的酶的亚基，其中硒是不稳定的，不存在硒代半胱氨酸。存在另一种可能性，即该蛋白质是硒结合/递送蛋白，其在硒代半胱氨酸的生物合成过程中或在硒掺入非硒代半胱氨酸硒蛋白如XD中时结合硒化合物。对这种蛋白质的进一步分离和研究将揭示细胞内硒处理的新信息。- 硒，黄嘌呤脱氢酶，梭菌属，钼羟化酶