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:? ? Selenophosphate synthetase (SPS) catalyzes the formation of selenophosphate from ATP and selenide. Selenophosphate is the selenium donor for the biosynthesis of selenocysteine-containing proteins, for the conversion of seryl-tRNA to selenocysteyl-tRNA, and for the synthesis of 2-selenouridine, a modified nucleoside present in tRNAs. Structural studies have been hampered by the difficulty in obtaining suitable crystals for x-ray crystallographic analysis. However, we have now successfully crystallized a mutant form (C17S) of SPS in which selenomethionine is substituted for methionine. The structure of this crystal has been determined and the results show that the SPS mutant exists as a homodimer with a flexible N-terminal region that consists of the first 20 amino acid residues. Furthermore, ATP binding to SPS is being investigated using a fluorescent ATP-derivative, trinitrophenyl-ATP, the wild type SPS, and two truncated SPS mutants. Preliminary results indicate that there are two ATP binding sites in each SPS molecule.? ? In an in vitro study, selenide in the millimolar range was used as the selenium substrate for SPS. This required level of selenide is highly toxic. Therefore, it is reasonable to assume the existence of a selenium-binding protein that could effectively deliver selenium to SPS at a much lower concentration of selenium. To this end, we investigated various potential candidates, including glyceraldehyde-3-phosphate dehydrogenase, a well-known glycolytic enzyme that has been shown to bind selenium from selenodiglutathione, presumably binding at the low pKa cysteine active site, and a novel selenium-binding protein isolated from Methanococcus vannielii that has been shown to bind inorganic selenium. This novel selenium-binding protein (SBP) has been cloned and expressed in E. coli. The recombinant protein consists of multiple subunits of the 8.8 kDa monomers and it migrates on native page gels as a ~42 kDa protein. SBP contains one cysteine, C59S, which exhibits a pK of ~6.7. With the purified protein, we have carried out structural studies using NMR spectroscopic methods. The initial results reveal that the monomeric structure of SBP consists of 1 alpha-helix and four beta-sheets. Crystallization of SBP is also in progress with the aim to obtain a suitable crystal for x-ray structural studies. In addition, to understand how selenium binds to SBP, we monitored the changes in protein mass using MALDI-TOF method. The results revealed the formation of a one-to-one selenium to 8.8 kDA SBP complex, which can be reversed by DTT treatment. This implies that selenium is bound to SBP via its cysteine residue. This notion is being confirmed using the SBP (C59S) mutant.? ? The interactions between SPS, SBP, and glyceraldehye-3-phosphate dehydrogenase were also investigated. Preliminary studies using selenium-bound SBP as the selenium donor and 32Pgamma-ATP for monitoring the SPS-catalyzed selenophosphate formation revealed that selenium-bound SBP was not a substrate for SPS. This observation indicates that SBP is not the proper selenium delivery protein for the E. coli SPS or additional factor(s) are required. In addition, the interaction between SPS and glyceraldehyde-3-phosphate dehydrogenase was studied using an affinity chromatographic method in which SPS was attached to CNBr-activated sepharose. The results indicate that glyceraldehyde-3-phosphate dehydrogenase binds to the immobilized SPS independent of the presence or absence of selenium.? ? Initial studies on selenium metabolism in a single-celled eukaryotic organism, the amoeba form of Dictyostellium, revealed the presence of about five selenium-containing proteins. Identification of these selenoproteins is in progress using mass spectrophotometric methods.
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