Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.
Selenium donor protein. It catalyzes the magnesium-dependent synthesis of a labile selenium donor compound from selenide and ATP (formation of mono-selenophosphate). selD gene product also is
essential for an entirely different process, a posttranscriptional tRNA modification involving the substitution of selenium for sulfur. This compound is required both for the incorporation of selenium into the wobble uridine-34 of several mnm5s2U-containing tRNAs and for the biosynthesis of selenocysteine from an L-serine residue ester bonded to unique tRNA-Ser (anticodon UCA). The exchange of sulfur in mnm5s2U-tRNA into mnm5se2U-tRNA is catalyzed by SelU. Certain Eukarya SelD-like protein lacks the selenide-dependent monophosphate synthetase activity and catalyses only the biosynthesis of selenocysteine.
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