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)磷酸硒是含硒半胱氨酸蛋白和硒- trna生物合成的硒供体。磷酸硒合成酶（SPS）催化ATP和硒化物生成磷酸硒。由于难以获得适合于x射线晶体学分析的晶体，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，其中硒代蛋氨酸取代了蛋氨酸。结构分析表明，SPS突变体以具有柔性n端区域的同二聚体存在。一种野生型SPS晶体也正在制备中，用于结构研究。体外动力学研究表明，SPS的硒化物Km为7.3微米，明显高于哺乳动物的毒性水平。因此，可以合理地假设存在一种硒传递蛋白，使得SPS可以在无毒的硒化物水平下发挥作用。为此，我们研究了多种潜在的候选蛋白，其中包括从万氏甲烷球菌中分离出的一种新的硒结合蛋白（SeBP）。为了更好地了解SeBP作为硒结合蛋白的作用机制，采用核磁共振（NMR）方法测定了SeBP的溶液结构。结果表明，SEBP以五聚体的形式存在，每个亚基由位于4链扭曲的γ -片上的γ -螺旋组成。五聚体结构主要通过疏水相互作用来维持，辅以氢键相互作用。令人惊讶的是，据信参与硒结合的半胱氨酸59位于靠近核心的柔性环中，不易被游离硒接触。此外，在该结构的指导下，位点定向突变发现Ile 9、Ser 22和Ile 25对于稳定五聚体结构至关重要。(b)含硒蛋白的过表达受到复杂的共翻译硒半胱氨酸结合机制的限制。为了解决这个问题，我们与彼得·舒尔茨和王江云合作，使用舒尔茨的方法在原核生物和真核生物中将一种非天然氨基酸结合到给定蛋白质的特定位点。该方法依赖于一个独特的密码子-tRNA对和相应的氨基酰基tRNA合成酶，用于不与宿主生物中任何内源性tRNA、氨基酰基tRNA合成酶、氨基酸或密码子交叉反应的非天然氨基酸。此外，我们还在开发一种硒蛋白表达系统，该系统使用Dictyostelium和Ed Korn博士获得的自主可复制质粒。(c)研究单细胞真核生物Dictyostelium阿米巴变形虫形式的硒代谢，我们发现亚硒酸盐处理在细胞生长过程中诱导了编码SelD、SelK和脱碘酶样蛋白的mRNA水平，亚硒酸盐和过氧化氢也诱导了编码硫氧还蛋白和glutaredoxin的mRNA水平。使用SelK基因敲除突变体的研究正在进行中。

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 as a homodimer with a flexible N-terminal region. A wild-type SPS crystal is also being prepared for structural studies. 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 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, among them a novel selenium-binding protein (SeBP) isolated from Methanococcus vannielii. To better understand how SeBP works as a selenium-binding protein, the solution structure of SeBP was determined by Nuclear Magnetic Resonance (NMR) methods. The results show that SEBP exists as a pentamer and each subunit is composed of a gamma-helix on top of a 4-stranded, twisted gamma-sheet. The pentameric structure is maintained mainly via hydrophobic interactions supplemented by hydrogen bond interactions. Surprisingly, cysteine 59, believed to be involved in selenium binding, is located in a flexible loop close to the core and not readily accessible to free selenium. In addition, site-directed mutagenesis, guided by the structure, revealed that Ile 9, Ser 22, and Ile 25 are essential for stabilizing the pentamer structure. (b) 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 and Jiangyun Wang to use Schultzs methodology to incorporate an 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 does not cross-react with any of the endogenous tRNAs, aminoacyl tRNA synthetases, amino acids, or codons in the host organism. In addition, we also are developing a selenoprotein expressing system using Dictyostelium and the autonomous replicable plasmid obtained from Dr. Ed Korn. (c) Investigating selenium metabolism in a single-celled eukaryotic organism, the amoeba form of Dictyostelium, we found that the mRNA levels, encoding SelD, SelK and an deiodinase-like protein, were induced by selenite treatment during cell growth, and selenite and hydrogen peroxide also induced mRNA levels encoding thioredoxin and glutaredoxin. Studies using the knockout mutant for SelK are in progress.