Fetal mouse selenophosphate synthetase 2 (SPS2): characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system.
A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect 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 mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.
Kim
I Y
IY
Laboratory of Biochemistry, National Heart, Lung, and Blood Institutes of Health, Bethesda, MD 20892, USA.
Guimarães
M J
MJ
Zlotnik
A
A
Bazan
J F
JF
Stadtman
T C
TC
D000263:Adenylate Kinase / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002412:Cations; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus; D015636:Magnesium Chloride / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D010770:Phosphotransferases; D011994:Recombinant Proteins; D018411:Spodoptera
D001426:Bacterial Proteins; D002412:Cations; D015226:Dinucleoside Phosphates; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D015636:Magnesium Chloride; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase; C499551:Sephs2 protein, mouse; C062762:selenophosphate synthetase