SPS catalyzes the exchange of the ATP gamma-phosphate group in the absence of selenide, suggesting that the beta-gamma-phosphate bond, but not the alpha-beta-phosphate bond, could be the initial cleavage site, formation of a phosphorylated enzyme intermediate, catalytic mechanism, overview
selenophosphate synthetase catalyzes the activation of selenide with ATP to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in tRNA anticodons
the phosphate moiety of selenophosphate is derived from the ATP gamma-phosphate group, while the orthophosphate is from the beta-phosphate group. Although Thr221 is not essential for the reaction, it contributes to the enzyme activity, probably by interacting with the ATP gamma-phosphate group and a water molecule. Sec/Cys13 and Lys16 are essential for the SPS catalysis. Thr221 and Gly222 interact with the gamma-phosphate group of ATP
selenophosphate synthetase catalyzes the activation of selenide with ATP to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in tRNA anticodons
the ATP-binding site is formed at the subunit interface of the homodimer. Four Asp residues coordinate four metal ions to bind the phosphate groups of AMPCPP. In the free SPS structure, the two loop regions in the ATP-binding site are not ordered, and no enzyme-associated metal is observed. This suggests that ATP binding, metal binding, and the formation of their binding sites are interdependent, Thr221 and Gly222 interact with the gamma-phosphate group of ATP
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant truncated mutant enzyme SPS-DELTAN, mixing of 0.001 ml of protein solution, containing 9.9 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, and 150 mM NaCl, with 0.001 ml of a reservoir solution containing 100 mM Na HEPES, pH 7.1, 50% 2-methyl-2,4-pentanediol, 200 mM ammonium phosphate, and 50 mM ammonium sulfate and equilibrating this mixture against 0.5 ml reservoir solution at 20°C, X-ray diffraction structure determination and analysis at 2.0 A resolution, hexameric crystal structure
SPS2 free or with bound alpha/beta-methylene ATP, X-ray diffraction structure determination and analysis at 1.98-2.1 A resolution, molecular replacement
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant trigger factor-fused SPS2 from Escherichia coli, during purification, the N-terminal trigger factor portion is removed by proteolysis with thrombin, and the final SPS protein comprises the full-length SPS region, residues 1-336, and nine extra N-terminal residues, GSGGIEGRH, which are derived from the linker
recombinant truncated mutant enzyme SPS-DELTAN from Escherichia coli by heat treatment at 70°C for 30 min, anion exchange and hydrophobic interaction chromatography, followed by another step of anion exchange chromatography and gel filtration