Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase.
ohr (organic hydroperoxide resistance gene) is present in several species of bacteria, and its deletion renders cells specifically sensitive to organic peroxides. The goal of this work was to determine the biochemical function of Ohr from Xylella fastidiosa. All of the Ohr homologues possess two cysteine residues, one of them located in a VCP motif, which is also present in all of the proteins from the peroxiredoxin family. Therefore, we have investigated whether Ohr possesses thiol-dependent peroxidase activity. The ohr gene from X. fastidiosa was expressed in Escherichia coli, and the recombinant Ohr decomposed hydroperoxides in a dithiothreitol-dependent manner. Ohr was about twenty times more efficient to remove organic hydroperoxides than to remove H(2)O(2). This result is consistent with the organic hydroperoxide sensitivity of Delta ohr strains. The dependence of Ohr on thiol compounds was ascertained by glutamine synthetase protection assays. Approximately two thiol equivalents were consumed per peroxide removed indicating that Ohr catalyzes the following reaction: 2RSH + ROOH --> RSSR + ROH + H(2)O. Pretreatment of Ohr with N-ethyl maleimide and substitution of cysteine residues by serines inhibited this peroxidase activity indicating that both of the Ohr cysteines are important to the decomposition of peroxides. C125S still had a residual enzymatic activity indicating that Cys-61 is directly involved in peroxide removal. Monothiol compounds do not support the peroxidase activity of Ohr as well as thioredoxin from Saccharomyces cerevisiae and from Spirulina. Interestingly, dithiothreitol and dyhydrolipoic acid, which possess two sulfhydryl groups, do support the peroxidase activity of Ohr. Taken together our results unequivocally demonstrated that Ohr is a thiol-dependent peroxidase.
Cussiol
José Renato Rosa
JR
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, São Paulo SP Brazil 05508-900.
Alves
Simone Vidigal
SV
de Oliveira
Marco Antonio
MA
Netto
Luis Eduardo Soares
LE
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics*; D020560:Proteobacteria / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D010544:Peroxidases; C058652:thiol-dependent peroxidase; D003545:Cysteine