Bacterial selenocysteine synthase--structural and functional properties.
Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.
Lehrstuhl für Mikrobiologie, Universität München, Germany.
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Tormay
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Wilting
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Lottspeich
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Mehta
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Christen
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Böck
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D019143:Evolution, Molecular; D006094:Gram-Positive Bacteria / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016999:Sulfur-Reducing Bacteria / Q000201:enzymology / Q000235:genetics*; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014166:Transferases; C060600:selenium transferase