Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue. Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase.
The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189--205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH(2)SO(2)F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH(2)SO(2)F. Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH(2)SO(2)F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD.
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
E
E
De Vendittis
T
T
Ursby
R
R
Rullo
M A
MA
Gogliettino
M
M
Masullo
V
V
Bocchini
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004274:DNA, Recombinant; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004274:DNA, Recombinant; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D013482:Superoxide Dismutase