FETAL MOUSE SELENOPHOSPHATE SYNTHETASE-2 (SPS2) - CHARACTERIZATION OFTHE CYSTEINE MUTANT FORM OVERPRODUCED IN A BACULOVIRUS-INSECT CELL SYSTEM

A novel gene detected in mouse embryonic sites of hematopoiesis was cl oned and shown to be a eukaryotic analog of the Escherichia coli selen ophosphate synthetase gene, Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is i ndicated by a TGA codon in the open reading frame of the gene in a pos ition corresponding to the essential cysteine of the E. coli enzyme, A n ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine containing enzyme a more active c atalyst, The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-inse ct cell system, Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme, The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide-dependent AMP formation from ATP, Although expression of wild-type enzyme has not been optimized, the mu tant form of the fetal mouse enzyme can be produced in amounts suffici ent for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cyst eine-containing prokaryotic enzyme.