Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.
Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.
Prouzet-Mauléon
Valérie
V
Laboratoires de Physiologie Moléculaire et Cellulaire, Bordeaux, France.
Monribot-Espagne
Christelle
C
Boucherie
Hélian
H
Lagniel
Gilles
G
Lopez
Sébastien
S
Labarre
Jean
J
Garin
Jérome
J
Lauquin
Guy J-M
GJ
D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D054464:Peroxiredoxins; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013434:Sulfenic Acids / Q000737:chemistry; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D020122:tert-Butylhydroperoxide / Q000737:chemistry
C515936:AHP1 protein, S cerevisiae; D013434:Sulfenic Acids; D006639:Histidine; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins; D013482:Superoxide Dismutase; C100870:cysteinesulfenic acid; D003545:Cysteine; D010100:Oxygen