Selenium Biochemistry

我们的研究集中在阐明硒蛋白在体内合成的基本机制，并研究硒酶的结构和功能。目前，我们正在调查以下项目： (A)硒磷酸盐是生物合成含硒半胱氨酸蛋白质和硒脱氧核糖核酸的供体。亚硒酸盐合成酶(SPS)催化三磷酸腺苷和亚硒酸盐生成亚硒酸盐。由于难以获得适用于X射线结晶学分析的晶体，SPS的结构研究一直受到阻碍。我们现在已经成功地结晶了一个SPS(C17S)突变体，其中硒蛋氨酸取代了蛋氨酸。结构分析表明，SPS突变体存在一个N-端区灵活的同源二聚体。一种野生型SPS晶体也正在准备进行结构研究。此外，体外动力学研究表明，SPS对硒的Km值为7.3微米，明显高于对哺乳动物的毒性水平。因此，合理地假设存在能够降低其Km值的组分或存在硒递送蛋白，以使SPS能够在无毒水平的硒的作用下发挥作用。为此，我们研究了各种可能的候选基因，包括已被证明与硒结合的著名糖酵解酶甘油醛-3-磷酸脱氢酶，以及从万尼氏甲烷球菌中分离的一种新的硒结合蛋白(SeBP)。这两种蛋白质都含有一个低pKA的半胱氨酸作为硒的结合部位。在这两种情况下，硒的供体是亚硒酸二谷胱甘肽，GSSeSG，GSH还原亚硒酸盐的中间体。为了更好地了解SeBP作为一种硒结合蛋白的作用机制，用核磁共振方法确定了SeBP的溶液结构。SeBP的单体是由四条β链形成的扭曲的β-折叠上的一个α-螺旋组成的。然而，在溶液中，SeBP形成稳定的五聚体。 (B)对单细胞真核生物-网茎基盘基菌的变形虫形式的硒代谢的初步研究表明，根据75-Se标记的蛋白质带的存在，大约存在3至4种含硒蛋白质。为了鉴定这些蛋白质，正在进行大规模的准备工作，以便为2D凝胶分离和蛋白质组学分析提供更大的蛋白质样本。 (C)含硒蛋白的过度表达受到复杂的共翻译硒半胱氨酸掺入机制要求的阻碍。为了绕过这个问题，我们与彼得·舒尔茨合作，使用他的方法将非天然氨基酸整合到原核生物和真核生物中给定蛋白质的特定位置。这种方法依赖于一对独特的密码子-tRNA和对应的非天然氨基酸的氨基酰基tRNA合成酶，这些非天然氨基酸不与宿主生物体中的任何内源tRNA、氨酰基tRNA合成酶、氨基酸或密码子发生交叉反应。最初，我们选择了三种硒蛋白--蛋氨酸亚麻酸还原酶B1、SelW蛋白和硫氧还蛋白还原酶1，因为我们可以通过监测最小的硒蛋白中的硒酶和SelW的比活性来验证合成产物的完整性。我们目前正在研究大肠杆菌表达系统。

Our research is focused on elucidating the basic mechanisms by which selenoproteins are synthesized in vivo and to investigate the structure and function of selenoenzymes. Currently, we are investigating the following projects: (a) Selenophosphate is the selenium donor for the biosynthesis of selenocysteine-containing proteins and seleno-tRNA. Selenophosphate synthetase (SPS) catalyzes the formation of selenophosphate from ATP and selenide. The structural study of SPS has been hampered by the difficulty in obtaining suitable crystals for x-ray crystallographic analysis. We have now successfully crystallized a SPS (C17S) mutant in which selenomethionine is substituted for methionine. The structural analysis revealed that the SPS mutant exists a homodimer with a flexible N-terminal region. A wild-type SPS crystal is also being prepared for structural studies. Furthermore, in vitro kinetic studies revealed a selenide Km of 7.3 microM for SPS, which is significantly higher than the toxic level for mammals. Therefore, it is reasonable to assume the presence of a component capable of lowering its Km value or the existence of a selenium delivery protein, such that the SPS can function with a non-toxic level of selenide. To this end, we have investigated various potential candidates, including glyceraldehye-3-phosphate dehydrogenase, a well-known glycolytic enzyme that has been shown to bind selenium, and a novel selenium-binding protein (SeBP) isolated from Methanococcus vannielii. Both proteins contain a low pKa cysteine as the selenium binding site. In both cases, the selenium donor is selenodiglutathione, GSSeSG, the intermediate in the reduction of selenite by GSH. To better understand how SeBP works as a selenium-binding protein, the solution structure of SeBP was determined by Nuclear Magnetic Resonnance (NMR) methods. The monomer of SeBP is composed of an alpha-helix on top of a twisted beta-sheet formed by four beta-strands. However, in solution, SeBP forms a stable pentamer. (b) Initial studies on selenium metabolism in a single-celled eukaryotic organism, the amoeba form of Dictyostelium, revealed the presence of about three to four selenium-containing proteins based on the presence of 75-Se-labeled protein bands. To identify these proteins, a large-scale preparation is being carried out to provide larger protein samples for 2D gel separation and proteomic analysis. (c) Overexpression of selenium-containing proteins is hampered by the requirement of a complex co-translational selenocysteine incorporation mechanism. To bypass this problem, we collaborated with Peter Schultz to use his methodology to incorporate unnatural amino acid into a specific site of a given protein in both prokaryotic and eukaryotic organisms. This method relies on a unique codon-tRNA pair and corresponding aminoacyl tRNA synthetase for the unnatural amino acid that do not cross-react with any of the endogenous tRNAs, aminoacyl tRNA synthetases, amino acids, or codons in the host organism. Initially, we chose three selenoproteins--methionine sulfoxide reductase B1, SelW protein, and thioredoxin reductase 1 since we can verify the integrity of the synthesized product by monitoring the specific activity of both selenoenzymes and SelW in the smallest selenoprotein. We are currently working with the E. coli expression system.