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J. Biol. Chem., Vol. 278, Issue 13, 11570-11578, March 28, 2003
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From the Departamento de Biologia, Instituto de Biociências,
Universidade de São Paulo, Rua do Matão 277, São Paulo SP Brazil 05508-900
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 H2O2. This result is consistent with the
organic hydroperoxide sensitivity of
Organic Hydroperoxide Resistance Gene Encodes a
Thiol-dependent Peroxidase*
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 + H2O.
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.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 55-11-30917589;
Fax: 55-11-30917553; E-mail: nettoles@ib.usp.br.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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