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 archa eon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expres sion in Escherichia coli obtained. The chemicophysical properties of the re combinant SsSOD were identical with those of the native enzyme. The recombi nant 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 fluor ide (PhCH2SO2F), a protease inhibitor routinely added during the preparatio n of cell-free extracts, showed that the modification was caused by PhCH2SO 2F. Refinement of the crystal model of SsSOD confirmed that a phenylmethane sulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH2SO2F behav ed as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modifi cation increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an importa nt role in the enzyme activity and the maintenance of the structural archit ecture of SsSOD.