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突变体作为具有柔性N-末端区域的同源二聚体存在。一个野生型SPS晶体也正在准备结构研究。体外动力学研究表明，SPS的硒化物Km为7.3 μ M，这明显高于对哺乳动物的毒性水平。因此，假设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，我们已经调查了各种潜在的候选人，其中一种新的硒结合蛋白（SeBP）从甲烷球菌vannielii分离。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。结果表明，SEBP以五聚体形式存在，每个亚基由4股扭曲的γ-折叠顶部的γ-螺旋组成。五聚体结构主要通过由氢键相互作用补充的疏水相互作用来维持。令人惊讶的是，被认为参与硒结合的半胱氨酸59位于靠近核心的柔性环中，并且不容易接近游离硒。此外，由结构指导的定点诱变揭示了Ile 9、Ser 22和Ile 25对于稳定五聚体结构是必需的。(b)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和Jiangyun Wang合作，使用Schultz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对和相应的氨酰tRNA合成酶，用于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的非天然氨基酸。此外，我们还正在开发一个硒蛋白表达系统，使用Dictyosteoprotein和从艾德科恩博士获得的自主复制质粒。(c)研究硒在单细胞真核生物中的代谢，阿米巴形式的网骨藻，我们发现，mRNA水平，编码SelD，SelK和脱碘酶样蛋白，在细胞生长过程中诱导亚硒酸盐处理，亚硒酸盐和过氧化氢也诱导mRNA水平编码硫氧还蛋白和谷氧还蛋白。使用SelK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.