Organic Hydroperoxide Resistance Gene Encodes a
Thiol-dependent Peroxidase*
José Renato Rosa
Cussiol,
Simone Vidigal
Alves,
Marco
Antonio de Oliveira, and
Luis
Eduardo Soares
Netto
From the Departamento de Biologia, Instituto de Biociências,
Universidade de São Paulo, Rua do Matão 277, São Paulo SP Brazil 05508-900
Received for publication, January 9, 2003
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ABSTRACT |
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
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.
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INTRODUCTION |
The infection of both plants and animals induces a defense
response that results in an oxidative burst with the increased generation of ROS1 (1). Lipid
hydroperoxides can be generated from the attack of ROS to the bacterial
membrane. Organic hydroperoxides can also be formed during metabolism
of certain drugs or during oxidation of n-alkanes (2). These peroxides
can then react with metals or with metalloproteins leading to the
production of secondary free radicals (3, 4), which may be related to
the fact that organic peroxides possess bactericidal activity (5).
The alkyl hydroperoxide reductase (AhpR) is frequently considered the
main enzyme responsible for the conversion of organic peroxides to the
corresponding alcohols in bacteria (6, 7). This enzyme comprises two
subunits, AhpF and AhpC. AhpC is a thiol-dependent peroxidase that belongs to the peroxiredoxin family (8). A cysteine
residue of AhpC is oxidized to sulfenic acid (R-SOH) by peroxides. NADH
reduces the sulfenic acid back to its sulfhydryl (R-SH) form in a
reaction catalyzed by AhpF. AhpF is a flavo-enzyme that shares homology
with thioredoxin reductase (9).
Recently a gene was isolated in Xanthomonas campestris
pv. phaseoli because its deletion rendered cells highly
sensitive to killing by organic peroxides but not to
H2O2 or superoxide generators (10). Therefore,
it was named organic hydroperoxide resistance (ohr) gene. ohr gene expression was highly
induced by t-bOOH, weakly induced by
H2O2, and not induced at all by superoxide
(10). Recently, homologues of this gene were also characterized in
other bacteria such as Bacillus subtilis and
Pseudomonas aeruginosa (11, 12) among others. Interestingly,
Ohr, but not AhpR, appears to play a significant role in Cu-OOH
resistance in B. subtilis (12). In Enterococcus
faecalis, ohr deletion rendered the cells more
sensitive to t-bOOH and also to ethanol (13). Sequence analysis has shown that ohr homologues are widely spread
among different bacteria genera, many of them pathogenic (14).
Ohr also shares similarities with OsmC, which is involved in bacterial defense against osmotic stress (14).
All the Ohr and OsmC homologues have two cysteine residues located in
motifs that are also very conserved. One of the cysteine residues is
part of a VCP motif that is also found in peroxiredoxins. Therefore, it
was postulated that Ohr could decompose peroxides directly, similarly
to AhpC, a peroxiredoxin found in bacteria (14). In fact, AhpC
complement ohr deletion in Escherichia coli and
in X. campestris (10). In P. aeruginosa, the
deletion of ohr rendered the cells more sensitive to organic
peroxide than ahpC deletion, and the double mutant
ohr, ahpC is more sensitive than the single
mutants (11). Finally, media from mutants for Ohr contain higher levels
of organic peroxides than the correspondent wild-type cells (11,
15).
Despite the suggestions that Ohr might directly detoxify organic
hydroperoxides, it was not possible to rule out the possibility that
Ohr is involved in other processes such as the transport of organic
molecules (10) or in yet undefined signaling pathways that lead to
activation of secondary molecules that would then inactivate organic
peroxides. Here, for the first time, the biochemical activity of Ohr
was elucidated: Ohr from Xylella fastidiosa possesses a
thiol-dependent peroxidase activity, which is probably
responsible for the hypersensitivity of
ohr mutants to
treatment with organic hydroperoxides.
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MATERIALS AND METHODS |
Materials--
All the reagents were purchased with the highest
degree of purity. DHLA was purchased in the reduced form from
Sigma (T8620). DHLA is a yellow oil, and its stock solution was
prepared by dilution to 50 mM concentration in 20 mM phosphate buffer, pH = 7.5 and heated at 45 °C
for 30 min. DHLA concentration was ascertained by the use of the
Ellman's reagent as described bellow.
Nucleic Acid Extraction, Cloning, and Nucleotide
Sequencing--
The ohr gene was PCR-amplified from the
cosmid XF-07F02 that was used in the X. fastidiosa
sequencing genome project (16). The following forward
5'-CGCGGATCCCATATGAATTCACTGGAG (Xfo1) and reverse
5'-CGCAAGCTTGGATCCTTAGTCAATCAG (Xfo2) primers were used. The underlined bases represent the NdeI and BamHI
sites, respectively. The PCR product was cloned into the pGEM-T easy
vector (Promega) resulting in the pGEM/ohr plasmid. An
E. coli DH5-
strain was transformed with
pGEM/ohr, and white colonies were selected from LB-ampicillin-5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal) medium. Plasmid extraction was performed using the Rapid Plasmid Miniprep System Concert kit (Invitrogen). The plasmid PGEM/ohr was used to generate the two individually ohr
mutant proteins, C61S and C125S, in which cysteines Cys-61 and Cys-125 were replaced by serines through PCR megaprimer methods (17, 18). In
the case of the C61S construct, PCR was performed first with the
mutagenic primer XfoC1 forward
5'-TTATTCTGCCTCTTTCATTGG-3' and Xfo2 reverse, where the bold
letters denote the mutation performed. A single band of 277 bp
was eluted out from the agarose gel and used as a primer (megaprimer)
along with the primer Xfo1 to the second PCR step to amplify the rest
of Ohr gene. In the case of the C125S construct, replacement
was performed in one-step PCR using the primers Xfo1 and a large
terminal mutagenic primer XfoC2 5'-CGCAAGCTTGGATCCTTAGTCAATCAGAATCAAAACGACGTCGATATTTCCACGGGTTGCATTAGAGTACGGAGAAACACGATGC-3', where the boldface typing represents the codon mutation and the underlined bases represents the BamHI restriction site. The
final mutated PCR products were ligated in pGEMT easy vector to produce PGEM/C61S and PGEM/C125S, independently.
pET15b, pGEM/ohr, PGEM/ohrC61S, and
PGEM/C125S were first digested with NdeI and
after by BamHI. The fragments generated by NdeI/BamHI digestion of plasmids derived from
pGEMs were extracted from agarose gel by the Rapid Gel Extraction
Concert kit (Invitrogen) and were individually ligated to the digested
pET-15b expression vector. The resulting pET15b/ohr,
PET15b/C61S, and PET15b/C125S plasmids were
sequenced in an Applied Biosystems ABI Prism 377 96 to confirm that the
constructions were correct. An E. coli DH5-
strain was
transformed with the expression vectors and cultured to increase
plasmid production. Another plasmid extraction was performed, and
E. coli Bl21(DE3) cells were transformed with the same
constructs. The resulting strains were used for expression and
purification of Ohr, C61S, and C125S.
Protein Expression and Purification--
E. coli
Bl21(DE3) strains transformed with pET15b/ohr,
pET15b/C61S, and pET15b/C125S were cultured (50 ml) overnight in LB + ampicillin medium, transferred to 1 liter of
fresh LB + ampicillin (100 µg/ml) medium, and cultured further
until the A600 reached 0.6-0.8.
Isopropyl-1-thio-
-D-galactopyranoside was then added to
a final concentration equivalent to 1 mM. After
3 h of incubation, cells were harvested by centrifugation. The
pellet was washed and suspended in the start buffer (phosphate buffer,
20 mM, pH 7.4). Seven cycles of 30 s of sonication
(35% amplitude) following 30 s in ice were applied to cell
suspension. The cell extracts were kept in ice during streptomycin
sulfate 1% treatment for 15 min. The suspension was centrifuged at
31,500 × g for 30 min to remove nucleic acid
precipitates. Finally cell extract was applied to a nickel affinity
column (Hi-trap from Amersham Biosciences). The conditions of protein
purification were optimized using the gradient procedure for imidazole
concentration described by the manufacturer.
Determination of Peroxide Concentration--
Peroxide
concentration was determined by the ferrous oxidation xylenol (FOX)
assay as previously described (3). Reactions were initiated by the
addition of thiol compounds and stopped at different intervals by
addition of 20 µl of HCl (1M) into 100-µl reaction
mixtures. No peroxide consumption was detected in the absence of
thiols. H2O2 concentration in stock solutions
was checked by its absorbance (
240 nm = 43.6 M
1·cm
1).
Determination of Sulfhydryl Groups Concentration--
The amount
of thiol groups remaining in solution was determined by the Ellman's
reagent (DTNB), using the
412 nm = 13,600 M
1·cm
1 for
2-nitro-5-thiobenzoic acid (TNB) (19). As described above, reactions
were stopped at different intervals by addition of 20 µl of HCl (1 M) into 100-µl reaction mixtures. Samples were
neutralized by dilution (1:10) in a solution containing Hepes (1 M, pH 7.4) and DTNB (5 mM). Absorbance at 412 nm was immediately recorded.
Glutamine Synthetase Protection Assay--
Antioxidant
activities of Ohr, OhrC61S, OhrC125S, and cTPxI were measured by their
ability to protect glutamine synthetase from oxidative inactivation.
Several H2O2-removing enzymes such as GSH
peroxidase, catalase, and cTPxI can protect glutamine synthetase (20).
The procedure used to determine glutamine synthetase activity was the
same described by Kim et al. (20).
Ohr Inactivation by NEM Treatment--
Recombinant Ohr (2 mg/ml)
was treated with NEM (1 mM) for 1 h at room
temperature and then was dialyzed against phosphate buffer (20 mM), pH 7.4. The concentration of histidine-tagged Ohr was
determined using the extinction coefficient
280 nm = 3960 M
1·cm
1, which was
determined using the software of ExPASy proteomics from Swiss
Institute of bioinformatics
(ca.expasy.org/tools/protparam.html).
Thrombin Proteolysis of Ohr--
Histidine tag of recombinant
Ohr was digested with thrombin (0.01 units/µl) using the thrombin
cleavage capture kit from Novagen. The digestion was carried out for
16 h at 25 °C. The concentration of the recombinant protein
without histidine tag was determined spectrophotometrically, using the
same extinction coefficient
280 nm = 3960 M
1·cm
1 determined above
because the histidine tag does not contain optically active residues.
Sulfenic Acid Formation--
Determination of sulfenic acid
(R-SOH) in wild-type as well as in mutant proteins was performed by the
TNB anion method described by Ellis and Poole (21). In summary, TNB was
prepared by incubation of an almost equimolar mixture of DTNB and DTT
(1 DTNB:0.9 SH). Proteins preincubated or not with peroxides were
treated with a 20-fold excess of TNB. As described before (21), TNB
reacts with sulfenic acids in a 1:1 stoichiometry, generating a mixed disulfide between TNB and a cysteine residue. Excess of TNB was removed
by PD-10 desalting column (Amersham Biosciences). Mixed disulfides were
then treated with 10-fold excess of DTT, and the amount TNB released
(which was equal to the amount sulfenic acid formed) was determined spectrophotometrically.
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RESULTS |
Genetic and Biochemical Analysis of Ohr--
Ohr from X. fastidiosa possesses a very high degree of similarity with
proteins from various bacteria such as X. campestris pv.
phaseoli and P. aeruginosa (14). In general, the
ohr gene is present in a single copy, but in some cases such
as B. subtilis, Mesorhizobium loti, and
Ralstonia solanacearum two copies of ohr are
present (12, 22, 23). In Streptomyces coelicolor, three copies of ohr appear to be present (24). A blastp analysis on the
X. fastidiosa genome using the tools available at the site aeg.lbi.ic.unicamp.br/xf/detected only one copy of ohr in
this bacteria located between coordinates 1,742,868 and
1,743,299 with 432 nucleotides. The predicted amino acid
sequence possesses 143 residues and a molecular mass equivalent to
14.9 kDa. Among other characteristics Ohr proteins have two
conserved cysteine residues that are at positions 61 and 125 in the
homologue from X. fastidiosa. Based on sequence homology two
domains of Ohr can be defined: domain 1, which contains cysteine 61 and
a high number of hydrophobic residues, and domain 2, which has cysteine
125 in a VCP motif (Fig. 1A).
The VCP motif is also present in the peroxiredoxin family, whose
proteins are thiol-dependent peroxidases (25). The two
proposed domains are highly conserved among Ohr homologues, especially
domain 1 (14) (Fig. 1A). Because ohr deletion
renders cells very sensitive to organic peroxide treatment, a
hydrophobicity analysis of Ohr protein was carried out (26). Our data
indicated that cysteine 61 is in a highly hydrophobic environment,
whereas cysteine 125 is in a hydrophilic environment (Fig.
1B).

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Fig. 1.
Domains of Ohr proteins that contain
cysteine residues. A, the sequences represented in this
scheme were the consensus obtained by the Clustal W method from the
MegAlign 5.01 software (DNAstar Inc.) including the Ohr proteins from:
Deinococcus radiodurans (Ohr Dr = GI:15806857);
Caulobacter crescentus (Ohr Cc GI:13422184);
Mesorhizobium loti (Ohr Ml1 = GI:14024371; Ohr Ml2 = GI:13475574); Vibrio cholerae (Ohr Vc = GI:15601759);
Mycoplasma genitalium (Ohr Mg GI:1723166); Mycoplasma
pneumoniae (Ohr Mp = GI:13508407); B. subtilis
(Ohr Bc1or YknA = GI:16078381, Ohr Bc2 = GI:16078379);
Staphylococcus aureus ssp. aureus Mu50 (Ohr
Sa = GI:15923818); Lactococcus lactis ssp.
lactis (Ohr Ll = GI:15672574); Listeria monocytogenes
EGD-e (Ohr Ll = GI:16804238); Listeria innocua
(Ohr Li = GI:16801366); S. coelicolor (Ohr Sc1 = GI:6562797, Ohr Sc2 = GI:7546676, Ohr Sc3 = GI:9885209);
Agrobacterium tumefaciens (Ohr At GI:15888188);
Sinorhizobium meliloti (Ohr Sm1 = GI:16263744, Ohr
Sm2 = GI:15964715); Bradyrhizobium japonicum (Ohr
Bj = GI:8708902); Acinetobacter calcoaceticus
(Ohr Ac = GI:7531260); Pseudomonas aeruginosa (Ohr
Pa = GI:15598046); Ralstonia solanacearum (Ohr Rs1 = GI:17549328, Ohr Rs2 = GI:17548547); Xanthomonas
campestris pv. phaseoli (Ohr Xc = GI:7531169);
Xylella fastidiosa (Ohr Xf = GI:15838425). The letters
in shade represent residues that match the consensus. The
histogram shows the strength of the residues belonging to the two
domains. B, hydrophilicity plot according to Ref. 26. In
this scheme, the amino acid sequence of Ohr from X. fastidiosa is displayed in the x-axis,whereas
hydrophilicity values are displayed in the y-axis. According
to this method, hydrophilic residues have positive values, whereas
hydrophobic residues have negative values. The arrows show
the positions of cysteines in Ohr sequence.
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To investigate whether Ohr possesses thiol-dependent
peroxidase activity, ohr gene from X. fastidiosa
was expressed in E. coli and purified by nickel affinity
chromatography (see "Materials and Methods"). A histidine tag
recombinant Ohr was obtained with high degree of purity as ascertained
by SDS-PAGE (Fig. 2). Two bands
corresponding to Ohr were observed, both of which migrate closely to 17 kDa, as expected for a monomer (Fig. 2A). Removal of
histidine tag by thrombin proteolysis also resulted in two bands both
of which migrated at ~15 kDa (data not shown). The abundance of each
band was dependent on the oxidative condition to which Ohr was exposed
(Fig. 2) and was independent of the presence of the histidine tag (data
not shown). Bands corresponding to dimers did not appear even after
treatments of Ohr with H2O2 or organic
hydroperoxide at various concentrations (Fig. 2B) indicating that no disulfide bond was formed between two Ohr molecules. Since the
two bands observed in Fig. 2 have approximately the monomer size, they
should represent different configurations of Ohr monomers. The lower
band (band a) should correspond to an oxidized
state of Ohr, which is generated after H2O2
treatment or at mild concentrations of t-bOOH (Fig.
2B). On the other side, the upper band (band
b) may represent a mixture of reduced and oxidized states of
Ohr. This is because Ohr treatment with increasing concentrations of DTT provoked augment of band b intensity (Fig.
2A) as well as Ohr treatment with high organic peroxide
concentrations (Fig. 2B). The meanings of these two bands
are further discussed below, after the description of results with Ohr
site-specific mutants. In any case, it is important to emphasize that
different monomeric configurations were also observed before for cTPxI,
a thiol-dependent peroxidase from Saccharomyces
cerevisiae (27).

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Fig. 2.
SDS-PAGE analysis of purified recombinant Ohr
from X. fastidiosa. Recombinant Ohr was expressed
in E. coli and purified as described under "Material and
Methods." Treatments were carried out for 1 h at room
temperature. A, lane 1, molecular mass standard
(BenchMarkTM Protein Ladder, Invitrogen); lane
2, untreated Ohr; lanes 3-6 represent Ohr treated with
the following concentrations of DTT: 60 µM, 1 mM, 10 mM, and 100 mM,
respectively. B, lane 1, molecular mass standard
(BenchMarkTM Protein Ladder, Invitrogen); lanes
2-5 represent Ohr treated with the following concentrations of
H2O2: 60 µM, 0.5 mM,
1 mM, and 10 mM, respectively; lanes
6-9 represent Ohr treated with the following concentrations of
t-bOOH: 60 µM, 0.5 mM, 1 mM, and 10 mM, respectively. Letters
a and b denote the lower and
upper band, respectively.
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Thiol-dependent Peroxidase Activity of
Ohr--
Recombinant Ohr decomposed peroxides only if DTT was also
present in the reaction mixture (Fig. 3).
Because the substrates were present in a 350-4100-excess over the
protein, it is reasonable to think that Ohr was acting catalytically.
In the conditions described in Fig. 3, the specific activity of Ohr was
20.0, 17.0, and 1.3 µM/min/ng when, respectively,
t-bOOH, Cu-OOH, and H2O2 were
considered the substrates. This indicates that Ohr was 10-20 times
more efficient in the removal of organic peroxides than in the removal
of H2O2. These results are consistent with the high susceptibility of
ohr mutants to organic peroxides
but not to H2O2 (Ref. 14 and references cited
herein). In any case, Ohr did decompose H2O2
when DTT was present in the reaction mixture. This ability of Ohr to
decompose peroxides was dependent on the integrity of its cysteine
residues because pretreatment of Ohr with NEM inhibited its peroxidase
activity (Fig. 3C). Therefore, it is reasonable to think
that Cys-61, Cys-125, or both are directly involved in the
decomposition of peroxides by Ohr.

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Fig. 3.
Thiol-dependent peroxidase
activity of Ohr. Peroxide concentration was determined at
different periods by FOX assay as described under "Material and
Methods." The reactions were carried out in Hepes buffer 50 mM, pH 7.4, in the presence of azide (1 mM) and
DTPA (0.1 mM). The concentration of peroxides at time zero
was 500 µM. Reactions were initiated by the addition of
DTT (0.5 mM). A represents the kinetics of
H2O2 decomposition in the presence of Ohr (100 ng/µl). B and C represent the kinetics of
Cu-OOH and t-bOOH decomposition, respectively, in the
presence of Ohr (10 ng/µl). represents the reaction mixture
without Ohr (DTT + peroxide); represents the full system (DTT + peroxide + Ohr). Reaction mixtures without DTT did not show any
decomposition of peroxides even in the presence of Ohr. The symbol in C represents Ohr whose cysteine residues were previously
alkylated with NEM.
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Ohr also possesses antioxidant property as demonstrated by its capacity
to protect glutamine synthetase from inactivation by thiol-containing
oxidative system composed of DTT/Fe3+/O2 or
composed of DHLA/Fe3+/O2 (Fig.
4A). Probably, this protection
was due to the thiol-dependent peroxidase activity of Ohr
because other thiol-dependent peroxidase such as GSH
peroxidase and cTPxI also protect glutamine synthetase from
inactivation (20). In fact, the glutamine synthetase protection assay
has been used to investigate whether proteins possess peroxide-removing activity (28, 29). Ohr did not protect glutamine synthetase against the
ascorbate/Fe3+/O2 system, indicating that this
protein is a thiol-specific antioxidant protein (Fig. 4A).
The protective activity of Ohr was dependent on the concentration of
protein and is comparable to the cTPxI activity (Fig. 4B).
Removal of histidine tag by thrombin treatment did not alter the
enzymatic characteristics of Ohr nor its ability to protect glutamine
synthetase (data not shown).

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Fig. 4.
Ohr protected glutamine synthetase from
oxidative inactivation. Glutamine synthetase (GS)
protection assay was performed as described under "Material and
Methods." All reaction mixtures contain: Fe3+ = 1 µM; glutamine synthetase 1 mg/ml; azide = 1 mM in Hepes buffer 50 mM, pH 7.4. A,
the symbols represent: (DTT 10 mM addition); (DTT
10 mM + Ohr 100 ng/µl addition); (ascorbate 10 mM addition); (ascorbate 10 mM + Ohr 100 ng/µl addition); (DHLA 10 mM addition); (DHLA 10 mM + Ohr 100 ng/µl addition). B, reactions
were carried out for 15 min. The symbols represent: = Ohr and = cTPxI at the concentrations described in the x-axis.
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When H2O2 was used as substrate, the specific
activity of Ohr (Fig. 3) was similar to cTPxI (data not shown). These
results are consistent with the glutamine synthetase assays (Fig.
4B) where these two proteins were also equally protective.
The oxidative inactivation of glutamine synthetase is dependent on
H2O2 formation by metal catalyzed oxidation
systems and by its posterior conversion to hydroxyl radical through the
Fenton reaction (30). Therefore protection of glutamine synthetase from
inactivation probably occurs through the removal of
H2O2 by antioxidant enzymes (31).
Analysis of Cysteine Replacements on Ohr Activity--
The role of
Ohr cysteines in peroxide reduction was strongly suggested since
treatment of Ohr with NEM lead to protein inactivation (Fig. 3). To
specifically investigate the roles of Cys-61 and Cys-125 on Ohr
catalysis these two residues were individually replaced by serine
generating, respectively, C61S and C125S. Initially, the capacity of
the mutant proteins to decompose hydroperoxides was investigated. C61S
and C125S had no detectable peroxidase activity when t-bOOH
was used as a substrate (Fig.
5A). When
H2O2 was the substrate, C125S had a residual
activity, whereas C61S did not decompose any peroxide (Fig.
5B). These results indicated that both cysteine residues are
important for catalysis. Replacement of either Cys-61 or Cys-125 also
provoked great decreases in the ability of Ohr to protect glutamine
synthetase from oxidative inactivation (Fig. 5C). C61S only
showed some protective effect at very high doses, which might be
attributable to nonspecific activity. Interestingly, replacement of two
of the cysteines of human peroxiredoxin V (prx V), which forms a stable
intramolecular disulfide intermediate during its catalytic cycle,
produced similar effects (32). Therefore, the results described in Fig.
5 represented an initial suggestion that an intramolecular disulfide is
also a reaction intermediate of wild-type Ohr. Further evidences are presented below.

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Fig. 5.
Effect of Ohr cysteine removal on peroxide
decomposition and on glutamine synthetase protection.
A, initial rate of t-bOOH decomposition by Ohr,
C125S, and C61S measured by the FOX assay. Protein concentrations were
2 ng/µl, and initial peroxide concentration was 200 µM.
Reactions were initiated by DTT (0.5 mM) addition.
B, initial rate of H2O2
decomposition by Ohr, C125S, and C61S measured by the FOX assay.
Protein concentrations were 50 ng/µl, and initial peroxide
concentration was 200 µM. Reactions were initiated by DTT
(0.5 mM) addition. C, glutamine synthetase
protection assay. All reaction mixtures contain: Fe3+ = 1 µM; glutamine synthetase 1 mg/ml; azide = 1 mM in Hepes buffer 50 mM, pH 7.4. Reactions
were carried out for 30 min. The symbols represent: Ohr, C125S,
C61S.
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The migration of Ohr mutants was also analyzed by SDS-PAGE (Fig.
6) under both reducing and non-reducing
conditions to understand the meaning of the two monomeric bands
observed in Fig. 2. As standards for mutant proteins, wild-type Ohr was
treated with peroxides (60 µM) and with DTT (100 mM), which provoked the appearance of the monomeric lower
(band a) and upper band (band
b) respectively. Interestingly, neither C61S nor C125S, in
any condition tested, migrated at the same position that the wild-type
protein treated with peroxides (60 µM) (band
a) (Fig. 6). Therefore, band a
observed for the wild-type protein (Fig. 2) should represent an
intramolecular intermediate since any of the mutants can form an
intramolecular disulfide bond. This was expected since an
intramolecular disulfide bond should lead to a more compact protein
configuration that would migrate faster than the other REDOX
states.

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Fig. 6.
Migration of mutant proteins in
SDS-PAGE. Treatments were carried out for 1 h. A,
lane 1, molecular mass standard (BenchMarkTM
Protein Ladder, Invitrogen); lanes 2-4 represent wild-type
Ohr treated with DTT (100 mM), t-bOOH (60 µM) and H2O2 (60 µM), respectively; lanes 5-7 represent C125S
treated with DTT (100 mM), t-bOOH (60 µM), and H2O2 (60 µM), respectively; B, lane 1,
molecular mass standard (BenchMarkTM Protein Ladder,
Invitrogen); lanes 2-4 represent wild-type Ohr treated with
DTT (100 mM), t-bOOH (60 µM), and
H2O2 (60 µM), respectively;
lanes 5-7 represent C61S treated with DTT (100 mM), t-bOOH (60 µM), and
H2O2 (60 µM),
respectively. Letters a, b, c, and
d refer to the bands described under "Results."
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Interestingly, two other bands, named c and d,
could also be observed in C125S exposed to oxidative conditions (Fig.
6A). Band d was detected in non-treated C125S
(data not shown) as well as in C125S submitted to mild oxidative
conditions (Fig. 6A, lane 7). Under stronger
oxidative conditions, band c became predominant and band d disappeared (Fig. 6A,
lane 6 and data not shown). Sometimes a band of ~30 kDa
was observed in substitution of band c after C125S treatment with organic peroxides (data not shown). The meaning of
band c is unknown and could be attributed to
proteolysis or to generation of cross-links in the dimer.
Since band d should correspond to a dimer of two
C125S proteins bound through their Cys-61, it appears that this
sulfhydryl group is relatively reactive toward peroxides. As expected
for a dimer exposed to reducing condition, when C125S was treated with
DTT in denaturing conditions, only a monomeric band (band b) was detected (Fig. 6A, lane 5).
On the contrary, C61S migrated preferentially as a monomer (Fig.
6B). Only after treatment of cells with very high peroxide concentrations could a dimer be observed (data not shown), indicating that Cys-125 is not very oxidizable by peroxides.
The possible formation of stable sulfenic acid intermediates (R-SOH) in
Ohr, C125S, and C61S was also analyzed. Using the compound TNB, we
could only clearly detect sulfenic acid intermediates in C125S protein
(Fig. 7). This result further indicated
that Cys-61 but not Cys-125 is very reactive.

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Fig. 7.
Sulfenic acid formation in C125S.
Sulfenic acid in C125S was measured by its reaction with TNB. C125S
(100 µM) with no pretreatment was incubated with TNB (4 mM) for 15 min at room temperature. A mixed disulfide
C125S-TNB was prepared as described under "Material and Methods."
Release of TNB from the mixed disulfide was recorded after 1, 3, 5, 10, and 30 min after addition of 10-fold excess of DTT, which corresponds
to the spectra 2-6. Spectrum 1 corresponds to the mixed disulfide
before DTT addition.
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Thiol Substrate Specificity--
The possibility that other thiol
compounds besides DTT support the peroxidase activity of Ohr was also
analyzed. No decomposition of t-bOOH by Ohr was detected
when DTT was replaced by monothiols such as GSH, 2-mercaptoethanol
(Fig. 8A), and cysteine (data
not shown). It is important to note that even when GSH was added at a
concentration 10-fold higher than DTT no peroxidase activity could be
observed (Fig. 8A). Therefore, Ohr does not possess GSH peroxidase activity.

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Fig. 8.
Thiol specificity of the Ohr peroxidase
activity. t-bOOH concentration was determined by the
FOX assay as described under "Material and Methods." Reactions were
initiated by addition of thiol compounds and terminated by addition of
20 µl of HCl (1 M) into 100-µl reaction mixtures.
Reactions were carried out in Hepes buffer 50 mM, pH 7.4 in
the presence of azide (1 mM) and DTPA (0.1 mM).
A, symbols represent the reactions with the following thiol
compounds: (GSH = 5 mM), (2-mercaptoethanol = 10 mM), (DTT = 0.5 mM), and (DHLA = 0.5 mM).
B, the symbols represent: (no further addition); (thioredoxin 80 ng/µl); (Ohr 5 ng/µl); and X (thioredoxin + Ohr).
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To check if thioredoxin could be the biological substrate for Ohr,
thioredoxin was added to the reaction mixture containing DTT,
t-bOOH, and Ohr. In the case of cTPxI, addition of
thioredoxin to the reaction mixture increased the specific activity of
this protein (28). The addition of thioredoxin from
Spirulina or from S. cerevisiae did not
increase significantly the ability of Ohr to decompose
t-bOOH, taking into account the decomposition of peroxides
by thioredoxin itself (Fig. 8B). The peroxidase activity of
Ohr may be specific for thioredoxin from X. fastidiosa.
Alternatively, other thiol compound than thioredoxin can be the
reducing agent of Ohr.
Interestingly, Ohr was also capable of decomposing peroxides (Fig.
8A) and protecting glutamine synthetase from inactivation (Fig. 4A) if DHLA was present in the reaction mixture. Like
DTT, DHLA is also a dithiol compound. Enzymes required for DHLA
biosynthesis are present in X. fastidiosa (XF1269, XF1270)
in an operon configuration indicating that this thiol compound should
be present in this bacteria. The possibility that DHLA is the in
vivo reducing power of Ohr is discussed below.
Because the ability of Ohr to decompose peroxides is dependent on the
presence of DTT, the stoichiometry of the reaction catalyzed by this
protein was investigated as described before for cTPxI (31). The
data described in Table I indicated that
the ratio of thiol consumption per peroxide consumption is around 2, which is consistent with the same reaction catalyzed by proteins
belonging to the peroxiredoxin family: 2RSH + ROOH
RSSR + ROH + H2O. Therefore, Ohr is a thiol-dependent
peroxidase.
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Table I
Stoichiometry of the reaction catalyzed by Ohr
Kinetics were started by addition of peroxide and were stopped by HCl
as described under "Materials and Methods." Assays for
determination of peroxide and sulfhydryl concentrations are also
described under "Materials and Methods."
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 |
DISCUSSION |
The present report attribute for the first time a biochemical
function for a protein belonging to the Ohr/OsmC family. Taken together, our results demonstrate unequivocally that Ohr from X. fastidiosa possesses thiol-dependent peroxidase
activity. This biochemical activity is consistent with the increased
sensitivity to organic peroxides observed for several bacterial species
in which this gene is deleted (10-13, 15).
Ohr possesses a very high specific activity for organic peroxides in
comparison with peroxiredoxins. In our hands, the specific activity of
Ohr is approximately 10-20 times higher than the specific activity of
cTPxI when organic peroxides were used as substrates (data not shown).
AhpC is the other thiol-dependent peroxidase present in
X. fastidiosa and in several other bacteria. AhpC belongs to
the peroxiredoxin family like cTPxI, and therefore they are expected to
behave similarly. Results showing that mutation of ohr
renders cells more sensitive to organic peroxide killing than ahpC deletion (11, 12) lead us to speculate that this
probably occurs because Ohr has higher specific activity toward organic peroxides than peroxiredoxins, but this suggestion awaits
experimental confirmation.
The relationship between Ohr and AhpCF proteins has been studied in
other bacteria. AhpC acts in concert with AhpF (a thioredoxin reductase
homologue) to reduce peroxides to the corresponding alcohols at the
expense of NADH (9). It is well known that the expression of
ahpC and ahpF are regulated by OxyR, a
transcriptional regulator that is activated by
H2O2 (33). This should also occur in X. fastidiosa because ahpC, ahpF, and
oxyR genes are contiguous and therefore probably belong to
the same operon (aeg.lbi.ic.unicamp.br/xf/). On the other hand, the
expression of ohr genes in B. subtilis and
X. campestris are not regulated by OxyR but by OhrR (12, 34). OhrR is a member of the MarR family of transcriptional repressors.
No OhrR homologue was found in a search through the site of X. fastidiosa genome suggesting that ohr is regulated by a
different mechanism in this microorganism.
X. fastidiosa contains several peroxide-removing
enzymes as analyzed by the bioinformatic tools available at
aeg.lbi.ic.unicamp.br/xf/and using the sequencing data generated by
the genome project supported by FAPESP (16). Besides ahpC
(XF1530), two other genes codify for proteins that contain
AhpC/TSA domains as defined by the pFAM analysis. Additionally,
one catalase and one GSH peroxidase homologue are also present. Each
one of these peroxide-removing enzymes may utilize different substrates
or may act during specific stress conditions.
Ohr is also capable of decomposing H2O2
although with a lower efficiency compared with the removal of organic
peroxides (Fig. 3). Probably Ohr does not play an important role in the
defense of X. fastidiosa against this oxidant. In other
related bacteria,
ohr mutants are not hypersensitive to
H2O2 (10, 11, 15). Moreover, in X. campestris pv. phaseoli and in B. subtilis
ohr expression is not regulated by OxyR, which is activated by
H2O2 (12, 34). Catalases appear to be the
primary defense of Xanthomonas against exogenous
H2O2 (35, 36). X. fastidiosa
possesses at least one catalase (XF2232), similar to HPI, (katG)
from E. coli, which is OxyR-regulated (33) and should be a
key component of the antioxidant defense against exogenous
H2O2. On the other side, several studies
indicate that AhpR should be an important component in the removal of
H2O2 endogenously generated in bacteria
(37-39).
The reduction of peroxides by Ohr requires at least one of its cysteine
residues because NEM pretreatment abolishes its peroxidase activity
(Fig. 3C). Ohr has two cysteine residues that are present in
all of its homologues (Fig. 1A) and therefore potentially
could be involved in peroxide reduction. Substitution of Cys-61 or
Cys-125 by serine dramatically reduces the ability of Ohr to decompose peroxides (Fig. 5), indicating that both cysteine residues are important for the catalytic activity. However, it is important to note
that C125S, but not C61S, still has a residual peroxidase activity,
suggesting an essential role for Cys-61. In fact, Cys-61, but not Cys
125, was easily oxidized by any of the peroxides (Fig. 6), and it
appears that Cys-61 is directly involved on peroxide reduction, whereas
Cys-125 is the resolving cysteine. Our data fit very well in the scheme
described in Fig. 9. At low
concentrations of organic peroxides, Cys-61 could be oxidized to
sulfenic acid, which should be rapidly converted to the intramolecular
disulfide intermediate (electrophoretic band a).
In support with this model, we could only detect sulfenic acid
intermediate in C125S protein (Fig. 7). Cys-61-SOH should be more
stable in C125S than in wild-type Ohr because the mutant protein lacks
Cys-125 to react with Cys-61-SOH. At high levels of organic peroxides,
sulfenic acid of Cys-61 should react first with another peroxide
molecule and not with Cys-125 sulfhydryl group, leading to the
formation of a cysteine sulfinic acid (R-SO2H). Further
reaction of Cys-61-SO2H with another organic peroxide
molecule can provoke the formation of Cys-61 sulfonic acid
(R-SO3H). Both Cys-61-SO2H and
Cys-61-SO3H should correspond to the electrophoretic
band b observed in Fig. 2 when Ohr was exposed to
high concentrations of organic peroxides.

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Fig. 9.
Proposed scheme for the enzymatic mechanism
of Ohr. The reduced form of Ohr (A) can react with
peroxides leading to the formation of Cys-61-SOH intermediate
(B), which can then be rapidly converted to an
intramolecular disulfide intermediate after reaction with Cys-125
(C). The intramolecular disulfide can be reduced back to
(A) by DTT or DHLA. In the presence of high amounts of
organic peroxides, Cys-61-SOH can be further oxidized to
Cys-61-SO2H (E) or Cys-61-SO3H
(F), which co-migrates with the reduced form of Ohr in the
position corresponding to the band b in the gels
of Figs. 2 and 6.
|
|
Disulfide intermediates are stable compounds among other factors
because they can not be overoxidized as sulfenic acids can be (40).
Therefore is tempting to speculate that Cys-125 prevents Ohr
inactivation by avoiding Cys-61 overoxidation to sulfinic or sulfonic
acids. It is well described that overoxidation of peroxiredoxin
provoked their inactivation (41). In the case of the mutant protein
C125S, in addition to overoxidation of Cys-61, dimer formation (Fig.
6A) could represent another pathway of protein oxidation.
Band b was never observed when Ohr was treated
with H2O2 (Fig. 2), indicating that this
peroxide has lower capacity than organic peroxides to overoxidize
Cys-61 to sulfinic or sulfonic acids. In fact,
H2O2 had lower ability than organic peroxides
to induce dimer formation in the mutant protein C125S (data not shown). Cys-61 is located in a very hydrophobic environment (Fig.
1B), which is probably ideal to accommodate an organic
peroxide but not H2O2. This hypothesis would
explain the higher specific activity of Ohr toward organic peroxides in
comparison with H2O2 (Fig. 3).
The biological-reducing substrate of Ohr is still unknown. GSH should
be present in X. fastidiosa because this bacteria contains homologues for the two genes (gsh1 and gsh2)
involved in its biosynthesis (aeg.lbi.ic.unicamp.br/xf/). However, this
thiol was not capable of reducing peroxides in the presence of Ohr,
even when it was present in a concentration ten times higher than DTT
concentration (Fig. 8A). Thioredoxin from neither
Spirulina (data not shown) nor from S. cerevisiae (Fig. 8B) increased the rate of peroxide removal by Ohr. We can not exclude, however, the possibility that thioredoxin or glutaredoxin systems of X. fastidiosa
specifically reduces Ohr.
In addition to DTT, DHLA supported the peroxidase activity of Ohr
(Figs. 4A and 8A). Therefore, Ohr utilized only
dithiols, but not monothiols, as substrate. Dithiols such as DTT and
DHLA have very negative redox potentials, which indicate that these compounds have very high reducing power. The redox potentials for
dithiols are in the range from
0.31 to
0.33, whereas for monothiols
such as GSH and cysteine the redox potentials are in the range of
0.24 to
0.25 (42). Probably the intramolecular disulfide bond of
Ohr is very stable, and only very strong reducing agents are able to
convert them to the reduced form. Another possibility is related to
possible structural constrains of the Ohr active site. According to our
results, the active site of this protein is very hydrophobic and may
not be capable of accommodating two monothiols but can interact with
only one dithiol molecule. This is because in the case of dithiols only
one molecule would be enough to reduce Ohr back to the dithiol
configuration according to Reaction 1.
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In the case of monothiols, two molecules would be required to
fully reduce Ohr. First, a mixed disulfide between Ohr and the
monothiol would be generated (Reaction 2), which would then be reduced
to the Ohr dithiol configuration by other monothiol molecule and the
release of a disulfide compound (Reaction 3).
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The possibility that DHLA is the biological substrate of Ohr is
supported by the fact that its biosynthetic pathway is present in
X. fastidiosa (XF 1269,XF1270). Interestingly, Bryk et
al. (43) characterized a peroxidase system dependent on lipoic
acid in Mycobacterium tuberculosis. Bryk et al.
(43) demonstrated that lipoic acid utilized came from a thiol linked
through an amide linkage dihydrolipoamide succinyltransferase, a
component of
-ketoacid oxidases, and was reduced by NADH in a
reaction catalyzed by dihydrolipoamide dehydrogenase. Both
dihydrolipoamide succinyltransferase (XF1549) and dihydrolipoamide
dehydrogenase (XF1548) enzymes are also present in X. fastidiosa (aeg.lbi.ic.unicamp.br/xf/). Several reducing systems
from X. fastidiosa are in the process to be expressed to
find the reducing substrate of Ohr. In any case, our data suggest
that Ohr may be a dihydrolipoic acid peroxidase.
Contrary to peroxiredoxins, GSH peroxidase, and catalases, Ohr belongs
to a family of proteins that are present only in bacteria, most of them
pathogenic to plants or mammals. Thus Ohr may be promising as a target
for drug development in agriculture and medicine, considering the fact
that plant and mammal defenses against pathogens involves generation of
oxidative burst (1).
 |
ACKNOWLEDGEMENTS |
We thank Gisele Monteiro for revising the
manuscript and Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) and Pró-Reitoria de Pesquisa
da Universidade de São Paulo for financial support.
 |
FOOTNOTES |
*
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.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M300252200
 |
ABBREVIATIONS |
The abbreviations used are:
ROS, reactive oxygen
Species;
AhpC, alkyl hydroperoxide reductase, subunit C;
AhpF, alkyl
hydroperoxide reductase, subunit F;
AhpR, alkyl hydroperoxide reductase
holo-protein;
DHLA, dyhydrolipoic acid;
DTNB, 5,5'-dithiobis(2-nitrobenzoic acid);
DTT, dithiothreitol;
FOX, ferrous
oxidation xylenol;
NEM, N-ethyl maleimide;
t-bOOH, tertbutylhydroperoxide;
Cu-OOH, cumene
hydroperoxide;
TNB, 2-nitro-5-thiobenzoic acid.
 |
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