Selenium Biochemistry

我们的研究重点是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，我们正在研究以下项目： (a)硒磷酸盐是含硒半胱氨酸蛋白质和硒tRNA生物合成的硒供体。 硒磷酸合成酶（SPS）催化ATP和硒化物形成硒磷酸。 SPS的结构研究一直受到难以获得合适的晶体进行X射线晶体学分析的阻碍。 我们现在已经成功地结晶SPS（C17 S）突变体，其中硒代蛋氨酸取代蛋氨酸。 结构分析表明，SPS突变体存在一个同源二聚体，具有一个灵活的N-末端区域。 一个野生型SPS晶体也正在准备结构研究。 此外，体外动力学研究显示SPS的硒化物Km为7.3 μ M，这显著高于对哺乳动物的毒性水平。 因此，假设存在能够降低其Km值的组分或存在硒递送蛋白是合理的，使得SPS可以在硒化物的无毒水平下起作用。 为此，我们研究了各种潜在的候选者，包括甘油三酸酯-3-磷酸脱氢酶，一种众所周知的糖酵解酶，已被证明可以结合硒，和一种新的硒结合蛋白（SeBP）分离自甲烷球菌vannielii。 两种蛋白质都含有低pKa半胱氨酸作为硒结合位点。 在这两种情况下，硒供体是硒二谷氨酸，GSSeSG，谷胱甘肽还原亚硒酸盐的中间体。 为了更好地理解SeBP作为硒结合蛋白的作用方式，通过核磁共振（NMR）方法确定SeBP的溶液结构。 SeBP的单体由位于由四条β链形成的扭曲β片层顶部的α螺旋组成。 然而，在溶液中，SeBP形成稳定的五聚体。 (b)在单细胞真核生物中硒代谢的初步研究，阿米巴形式的网骨藻，揭示了存在约三至四个含硒蛋白的基础上存在的75-Se标记的蛋白带。 为了鉴定这些蛋白质，正在进行大规模制备，以提供更大的蛋白质样品用于2D凝胶分离和蛋白质组学分析。 (c)含硒蛋白的过表达受到复杂的共翻译硒代半胱氨酸掺入机制的要求的阻碍。 为了绕过这个问题，我们与Peter Schultz合作，使用他的方法将非天然氨基酸掺入原核和真核生物中给定蛋白质的特定位点。 该方法依赖于独特的密码子-tRNA对和相应的非天然氨基酸的氨酰tRNA合成酶，其不与宿主生物体中的任何内源性tRNA、氨酰tRNA合成酶、氨基酸或密码子交叉反应。 最初，我们选择了三个硒蛋白-甲硫氨酸亚砜还原酶B1，SelW蛋白和硫氧还蛋白还原酶1，因为我们可以通过监测硒酶和SelW在最小的硒蛋白中的比活性来验证合成产物的完整性。 目前，我们正在与E。coli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.