Study of Selenium-Dependent Xanthine Dehydrogenase and Purine Hydroxylase

硒依赖性黄嘌呤脱氢酶和嘌呤羟化酶的研究

• 批准号: 6432635
• 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/6432635
• 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 recently been gained 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 (XDH) from Clostridium. Selenium present in NAH is somewhat labile and can be removed by treatment with chaotropic agents, such as sodium dodecyl sulfate. In order to better understand the nature of selenium and the incorporation of selenium, XDH from Clostridium purinolyticum was purified to homogeneity. XDH was found to consist of three subunits (based on SDS-PAGE) and to be labeled with selenium (75-Se). This selenium was labile and presumably is not in the form of selenocysteine. During the isolation of this enzyme, another selenium-containing hydroxylase was isolated and termed purine hydroxylase (PH). PH consisted of four subunits (based on denaturing gel electrophoresis) and utilized purine and hypoxanthine as substrates. Selenium in PH was also labile, and thus represents yet another non-selenocysteine selenium-requiring enzyme to be characterized and to be used in a comparative analysis of these unique enzymes. Now that a good purification scheme has been determined for the isolation of both XDH and PH from C. purinolyticum, these enzymes will be investigated in more detail. These investigations will focus on the identification of cofactors and metals present, as well as study of the active sites for enzyme reaction and electron transfer using methods such as electron paramagnetic resonance (EPR) and x-ray absorption spectroscopy (XAS). The focus of the investigation will revolve around determining the moiety of selenium present in the active site and its relationship to other cofactors (e.g. molybdenum cofactor). Once more knowledge is gained into the properties of selenium in these enzymes, the mechanism by which the cell incorporates selenium into this active site will be investigated. This work may uncover a new delivery protein(s) or selenium donor molecule required for XDH and PH and possibly other molybdenum hydroxylases, and perhaps shed light on the delivery of selenium in the biosynthesis of selenocysteine.

虽然最近已经获得了显着的知识硒纳入硒蛋白，这项工作的重点是放在酶，携带硒的形式硒半胱氨酸。 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（XDH）。 存在于NAH中的硒是稍微不稳定的，并且可以通过用离液剂（chaotropic agents）（例如十二烷基硫酸钠）处理来去除。 为了更好地了解硒的性质和硒的掺入，将来自解嘌呤梭菌的XDH纯化至均一。 发现XDH由三个亚基组成（基于SDS-PAGE），并且被硒（75-Se）标记。 这种硒是不稳定的，可能不是硒代半胱氨酸的形式。 在分离这种酶的过程中，分离出另一种含硒羟化酶，并命名为嘌呤羟化酶（PH）。 PH由四个亚基组成（基于变性凝胶电泳），并利用嘌呤和次黄嘌呤作为底物。 硒在PH值也是不稳定的，因此代表了另一种非硒代半胱氨酸硒需要酶的特点，并用于这些独特的酶的比较分析。目前已确定了从C.因此，将对这些酶进行更详细的研究。 这些研究将集中在辅因子和金属的鉴定，以及使用电子顺磁共振（EPR）和X射线吸收光谱（XAS）等方法研究酶反应和电子转移的活性位点。 研究的重点将围绕确定活性位点中存在的硒部分及其与其他辅因子（例如钼辅因子）的关系。 一旦对这些酶中硒的性质有了更多的了解，将研究细胞将硒结合到这个活性位点的机制。 这项工作可能揭示XDH和PH以及可能的其他钼羟化酶所需的新的递送蛋白或硒供体分子，并可能阐明硒在硒代半胱氨酸生物合成中的递送。