Ohr plays a central role in bacterial responses against
fatty acid hydroperoxides and peroxynitrite
Thiago G. P. Alegriaa,1
, Diogo A. Meirelesa,1
, José R. R. Cussiola,2
, Martín Hugob,3
, Madia Trujillob
, Marcos Antonio de
Oliveirac
, Sayuri Miyamotod
, Raphael F. Queirozd,4
, Napoleão Fonseca Valadarese,5
, Richard C. Garratte
, Rafael Radib,6
,
Paolo Di Masciod
, Ohara Augustod
, and Luis E. S. Nettoa,6
a
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, 05508-090, Sao Paulo, Brazil; b
Departamento de
Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay;
c
Departamento de Biologia, Universidade Estadual Paulista Júlio de Mesquita Filho, Campus do Litoral Paulista, 11330-900, Sao Vicente, Brazil;
d
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil; and e
Instituto de Física de São Carlos,
Universidade de São Paulo, 13566-570, Sao Carlos, Brazil
Contributed by Rafael Radi, November 30, 2016 (sent for review October 8, 2016; reviewed by Leopold Flohé and Leslie B. Poole)
Organic hydroperoxide resistance (Ohr) enzymes are unique Cysbased,
lipoyl-dependent peroxidases. Here, we investigated the
involvement of Ohr in bacterial responses toward distinct hydroperoxides.
In silico results indicated that fatty acid (but not cholesterol)
hydroperoxides docked well into the active site of Ohr from Xylella
fastidiosa and were efficiently reduced by the recombinant enzyme
as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay.
Indeed, the rate constants between Ohr and several fatty acid hydroperoxides
were in the 107
–108
M−1
·s−1
range as determined by a
competition assay developed here. Reduction of peroxynitrite by
Ohr was also determined to be in the order of 107
M−1
·s−1
at pH 7.4
through two independent competition assays. A similar trend was
observed when studying the sensitivities of a Δohr mutant of Pseudomonas
aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides,
which are readily solubilized by bacterial surfactants,
killed the Δohr strain most efficiently. In contrast, both wild-type and
mutant strains deficient for peroxiredoxins and glutathione peroxidases
were equally sensitive to fatty acid hydroperoxides. Ohr also
appeared to play a central role in the peroxynitrite response, because
the Δohr mutant was more sensitive than wild type to 3-morpholinosydnonimine
hydrochloride (SIN-1 , a peroxynitrite generator). In
the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole
(a catalase inhibitor) were the most sensitive. Furthermore, fatty acid
hydroperoxide and SIN-1 both induced Ohr expression in the wildtype
strain. In conclusion, Ohr plays a central role in modulating the
levels of fatty acid hydroperoxides and peroxynitrite, both of which
are involved in host–pathogen interactions.
hydroperoxides | thiols | Cys-based peroxidase | pathogenic bacteria |
Pseudomonas aeruginosa
Thiol peroxidases are receiving increasing attention as biological
sensors and signal transducers of hydroperoxide-dependent
pathways (1–3). Peroxiredoxin (Prx) and glutathione peroxidase
(Gpx) are central players in hydroperoxide metabolism and sensing,
which is consistent with their high overall abundance and reactivity
(3).
The issue of substrate specificity in Cys-based peroxidases is
intriguing. Prx enzymes are highly reactive to hydroperoxides but
not to other oxidants or thiol reagents, such as chloroamines or
iodoacetamide (4). The structural features responsible for this
phenomenon are still emerging (5–7), including some more subtle
aspects of their specificity. For instance, the three different Prxs
from Mycobacterium tuberculosis display distinct hydroperoxide
specificities with alkyl hydroperoxide reductase E (AhpE) being
highly specialized toward long-chain fatty acid hydroperoxides (8).
Furthermore, yeast Tsa1, yeast Tsa2, rat Prx6, and human Prx2 are
more reactive toward H2O2 (9–11), whereas human Prx5 is more
reactive toward organic hydroperoxides and peroxynitrite (12, 13).
Organic hydroperoxide resistance (Ohr) and osmotically inducible
protein C (OsmC) form another group of Cys-based
peroxidases that are biochemically and structurally distinct from
Prx and Gpx. Ohr was first described in Xanthomonas campestris,
through the characterization of a null mutant strain (Δohr). The
Δohr strain displays high sensitivity toward tert-butyl hydroperoxide
(t-BHP), but not to H2O2. Moreover, transcription of the
ohr gene is specifically induced by t-BHP (14). Later on, this
enzyme was found in several bacteria (15). Subsequently, we and
others were able to show that this “organic hydroperoxide resistance”
phenotype is due to the Cys-based, thiol-dependent
peroxidase activity of Ohr (16, 17). In contrast, OsmC proteins
were initially implicated in the osmotic stress response (18), and
only later were shown to share structural and biochemical similarities
with Ohr (19, 20). Ohr/OsmC enzymes present an
α/β-fold that is very distinct from the thioredoxin-fold characteristic
of Prx and Gpx (17, 21, 22). Furthermore, Ohr/OsmC
Significance
Hydroperoxides play central roles in cell signaling. Hydroperoxides
of arachidonic acid are mediators of inflammatory processes in
mammals, whereas hydroperoxides of linoleic acid play equivalent
roles in plants. Peroxynitrite is also involved in host–pathogen
interactions, and hydroperoxide levels must therefore be strictly
controlled by host-derived thiol-dependent peroxidases. Organic
hydroperoxide resistance (Ohr) enzymes, which are present in
many bacteria, display unique biochemical properties, reducing
fatty acid hydroperoxides and peroxynitrite with extraordinary
efficiency. Furthermore, Ohr (but not other thiol-dependent peroxidases)
is involved in the Pseudomonas aeruginosa response to
fatty acid hydroperoxides and to peroxynitrite, although the latter
is more complex, probably depending on other enzymes. Therefore,
Ohr plays central roles in the bacterial response to two hydroperoxides
that are at the host–pathogen interface.
Author contributions: T.G.P.A., D.A.M., J.R.R.C., M.H., M.T., S.M., R.R., P.D.M., O.A., and
L.E.S.N. designed research; T.G.P.A., D.A.M., J.R.R.C., M.T., M.A.d.O., S.M., R.F.Q., and N.F.V.
performed research; M.H. and M.T. contributed new reagents/analytic tools; T.G.P.A., D.A.M.,
J.R.R.C., M.T., R.C.G., R.R., O.A., and L.E.S.N. analyzed data; and T.G.P.A., D.A.M., M.T., R.C.G.,
R.R., and L.E.S.N. wrote the paper.
Reviewers: L.F., University of Padua; and L.B.P., Wake Forest School of Medicine.
The authors declare no conflict of interest.
1
T.G.P.A. and D.A.M contributed equally to this work.
2
Present address: Departamento de Bioquímica, Instituto de Química, Universidade de
Sao Paulo, 05508-000, Sao Paulo, Brazil.
3
Present address: German Institute of Human Nutrition, 14558 Potsdam, Germany.
4
Present address: Departamento de Ciências Naturais, Universidade Estadual do Sudoeste
da Bahia, Vitória da Conquista, 45083-900, Bahia, Brazil.
5
Present address: Laboratório de Biofísica Molecular, Departamento de Biologia Celular,
Universidade de Brasília, CEP 70910-900, Brasília, Brazil.
6
To whom correspondence may be addressed. Email: rradi@fmed.edu.uy or nettoles@ib.
usp.br.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1619659114/-/DCSupplemental.
E132–E141 | PNAS | Published online December 27, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1619659114
enzymes accept electrons from lipoyl groups of proteins (23, 24)
in contrast to Prx and Gpx, which are most commonly reduced by
thioredoxin or glutaredoxin/glutathione (GSH) (1, 2).
Despite all of these studies, the identification of the natural
oxidizing substrates of Ohr is still a challenge. It is known that the
Ohr from Xylella fastidiosa (XfOhr) is 10,000-fold more efficient in
the removal of t-BHP compared with H2O2 (23). However, because
t-BHP and cumene hydroperoxide (CHP) are artificial
compounds, the identity of the biological oxidizing substrates for
Ohr remains to be identified. We previously raised the hypothesis
that elongated hydrophobic molecules, such as hydroperoxides
derived from fatty acids, could be the natural substrates, because
these compounds fitted very well into the electron density corresponding
to a PEG molecule found bound to the active site of Ohr
in the crystal structure (22). Other corroborating evidence came from
the observation that the Ohr and its transcriptional repressor (OhrR)
system are responsible for linoleic acid hydroperoxide (LAOOH)
clearance in Xanthomonas spp. (25). Furthermore, LAOOHs induce
ohr transcription, at higher levels than t-BHP in some cases (25–27).
Here, we systematically investigated the involvement of Ohr in
the bacterial response toward distinct hydroperoxides. Initially,
using different approaches, we showed that XfOhr preferentially
reduced long-chain fatty acid hydroperoxides and also peroxynitrite.
Furthermore, microbiological assays using Pseudomonas
aeruginosa correlated well with the biochemical/kinetic assays.
Taken together, our results indicated that Ohr protein is central
to the response of the bacterium to fatty acid hydroperoxides and
peroxynitrite, although the process appears to be more complex
in the latter case, possibly involving the cooperation of other
enzymes. In contrast, catalase is a central player in the response
of P. aeruginosa to exogenously added H2O2. The experimental
approach presented herein allows defining the relative sensing
and detoxifying roles of various peroxidatic systems in bacteria
challenged with different biologically relevant hydroperoxides.
Results
Fatty Acid Hydroperoxides Dock into the Ohr Active Site. Previous
data suggested that the active site cavity of XfOhr preferentially
accommodates elongated molecules (22). Motivated by this observation,
we performed an unbiased docking analysis to gain
further insight into the affinity of Ohr protein for different organic
hydroperoxides. The full structure of XfOhr [Protein Data Bank
(PDB) ID code 1ZB8] was analyzed against all cis and trans isomers
of hydroperoxides derived from mono- or polyunsaturated
fatty acids (Fig. 1 A and C).
Long-chain fatty acid hydroperoxides docked well into the
XfOhr active site, overlapping with the position occupied by a
PEG molecule in the Ohr crystal structure. All of the elongated
hydroperoxide molecules produced good fits with their O–O bond
pointing toward the catalytic cysteine (Cys61
, so-called peroxidatic
Cys), although trans isomers gave higher scores (Fig. 1 B and D).
However, it was not possible to observe any preferred orientation
of the carboxyl group in relation to the surface of the enzyme,
suggesting that hydrophobic interactions are major factors for the
binding and orientation of the hydroperoxide within the active site
(Fig. 1 and Fig. S1A). This finding is consistent with the fact that no
systematic difference could be observed between S and R enantiomers.
In the case of hydroperoxides containing more than one
peroxidation, no preference for the peroxide group pointing toward
Cys61
was detected. However, in stark contrast, neither the cis
nor trans isomers of cholesterol hydroperoxide (ChOOH) docked
well into the active site cavity (Fig. S1B). For example, the fitness
scores for the docked ChOOHs, which varied between 30.1 and
47.1, were, on average, 38% lower than the fitness scores for hydroperoxides
derived from arachidonic acid (54.8–67.3). This
finding is all the more significant, given that the cholesterol derivatives
contain more atoms; therefore, the ligand efficiency for
these molecules would be even lower. For the cholesterol derivatives,
the lower fitness values are probably due to the failure to
orient the hydroperoxide moiety adequately toward the catalytic
cysteine and/or to their rigidity, which prohibits optimizing hydrophobic
interactions within the binding site cavity. These data support
the hypothesis that elongated hydrophobic molecules are the
preferred oxidizing substrates of Ohr proteins (22).
Biochemical Characterization of Peroxidase Specificity. Next, we tested
the ability of XfOhr to reduce various lipid-derived hydroperoxides
to validate the docking analyses. Thiol peroxidase activity was
assessed using the previously described lipoamide-lipoamide dehydrogenase–coupled
assay (23). In contrast to other thioldependent
peroxidases, lipoylated proteins (but not thioredoxin or
glutathione) are the most probable physiological reductants of
XfOhr and osmotically inducible protein C from Escherichia coli
(EcOsmC) (23). However, alternative ways of Ohr reduction (e.g.,
redoxins) cannot be excluded and should be tested in the future.
Following the reduction of an artificial organic hydroperoxide
(t-BHP) and applying a bisubstrate steady-state approach, it
was previously determined that Ohr displays a ping-pong kinetic
mechanism, and the true Km values for dihydrolipoamide and lipoylated
proteins are in the 10–50 μM range (23).
A major challenge here was to establish conditions for solubilizing
hydrophobic hydroperoxides without compromising the
Fig. 1. Molecular docking of long-chain hydroperoxides into the Ohr active
site pocket. The molecular surface of XfOhr (PDB ID code 1ZB8) is represented
in brown, whereas the hydroperoxides are shown as lines with the
atoms colored as follows: carbon = purple (A and B) and cyan (C and D),
oxygen = red, and hydrogen = white. (A and C) Results of docking simulations,
showing the entire protein structure. Ligands used hydroperoxides
derived from OAOOH, palmitoleic acid hydroperoxide, and LAOOH (A) and
arachidonic acid hydroperoxide (C): 5-HpETE, 5(S)-HpETE, 8-HpETE, 9-HpETE,
12-HpETE, 15-HpETE, and 15(S)-HpETE. Oleic and linoleic acid molecules
containing the hydroperoxide group in both the cis- and trans-configurations
or in different combinations of the two (linoleic hydroperoxides) were
considered in the analysis. (B and D) Details of the Ohr active site pocket,
showing the hydroperoxide groups of the fatty acid hydroperoxides facing
toward the Sγ of Cys61
. The atoms of the enzyme are represented as spacefilling
spheres and colored brown, except for the sulfur atom of Cys61
(yellow).
The docking procedures were performed using Gold software (70), and molecular
graphics were generated using PyMOL (www.pymol.org/).
Alegria et al. PNAS | Published online December 27, 2016 | E133
BIOCHEMISTRYPNASPLUS
structure and activity of XfOhr. Initially, XfOhr activity was evaluated
in the presence of a series of detergents and organic solvents
(Fig. S2A), using a highly water-soluble organic hydroperoxide,
t-BHP, as a substrate. XfOhr retained more than 80% of its original
activity toward t-BHP when Tween-20 (0.25%) was used. At the
same time, the activity toward LAOOH was maximal (Fig. S2B),
indicating good solubilization of LAOOH (100 μM). Therefore,
Tween-20 was chosen to solubilize hydrophobic hydroperoxides in
the lipoamide-lipoamide dehydrogenase–coupled assays.
Reduction of LAOOH by XfOhr presented a complex steadystate
kinetic behavior that was biphasic: At low concentrations, up
to 20 μM, the rate of the reaction increased hyperbolically with
increasing concentrations of LAOOH, as expected for a Michaelis–
Menten model (Fig. 2A). However, above 20 μM, the rate dropped
markedly as a function of LAOOH concentration (Fig. 2A).
Considering only the first phase (up to 20 μM), we estimated
by nonlinear regression a kcat app of 36 s−1
and a Km app of 7.3 μM
at 50 μM dihydrolipoamide (Fig. 2A, Inset), which indicated that
XfOhr is highly efficient in the removal of LAOOH (kcat app/Km app =
5 × 106
M−1
·s−1
). However, one major limitation of this analysis
was that saturation was not achieved; therefore, the kinetic parameters
have probably been underestimated. Consistent with this
result, we note that data for Ohr from Mycobacterium smegmatis
also did not fit well to the Michaelis–Menten model at high
LAOOH concentrations; however, in this case, kcat app/Km app was
estimated to be in the range of 105
M−1
·s−1
(24).
The catalytic efficiency for LAOOH was in the same range as
the range previously determined for t-BHP, considering true kcat
and Km values in the latter case (23). Unfortunately, the biphasic
shape of the curves (Fig. 2A) does not allow us to obtain true
enzymatic parameters for fatty acid hydroperoxides. Therefore,
it was not possible to compare the reduction of fatty acid and
artificial organic hydroperoxides on the same grounds.
We also evaluated the possibility that XfOhr could reduce hydroperoxides
derived from unsaturated fatty acids attached to
phosphatidylcholine, which could reflect the capacity of the enzyme
to reduce hydroperoxides resident in membranes, similar to the
activity described for phospholipid Gpx4 (28, 29). However, no
such activity for XfOhr was detected, although it was capable of
reducing both t-BHP and LAOOH in the same experiment (Fig.
2B). Moreover, when phosphatidylcholine hydroperoxide was incubated
with phospholipase A2 (PLA2), releasing the corresponding
free fatty acid hydroperoxide, NADH consumption was
observed (Fig. 2C). Therefore, it appears that fatty acid hydroperoxides
attached to phosphatidylcholine were not accessible to
the XfOhr active site. Furthermore, under the same conditions,
XfOhr did not reduce ChOOH (Fig. 2B), consistent with the
docking analysis. In these experiments, Triton X-100 (0.15%) was
used instead of Tween-20 (0.25%) to solubilize ChOOH, because
this procedure results in good solubilization of this lipid (30). The
products of oleic hydroperoxide (OAOOH) and LAOOH reduction
by XfOhr were confirmed to be the corresponding alcohols
by mass spectrometry (Fig. S3).
Regarding the non-Michaelis–Menten behavior of XfOhr at
substrate concentrations higher than 20 μM (Fig. 2A), two nonexclusive
hypotheses can be proposed: micelle formation of lipid
hydroperoxide molecules and/or hyperoxidation of the peroxidatic
cysteine of XfOhr (Cys61
) to sulfinic acid (Cys-SO2H−
) or sulfonic
acid (Cys-SO3H−
) (Fig. 3A).
Considering the first hypothesis, the critical micelle concentration
(CMC) lies in the range of 6–60 μM for most unsaturated fatty
acids, depending on pH and the counter-cation used (31–34),
which is consistent with the inflection point of 20 μM (Fig. 2A).
However, Tween-20 was present at a 0.25% concentration, which
appears to solubilize LAOOH, at least up to a 100-μM concentration
(Fig. S2B).
So, we investigated if hyperoxidation could be involved in the
second phase decay (Fig. 2A). Among Cys-based peroxidases, it is
well known that sulfenic acid (Cys-SOH) formed in the peroxidatic
Cys can have two fates: (i) disulfide formation through a condensation
reaction toward a second Cys residue or (ii) hyperoxidation
to sulfinic acid and sulfonic acid due to a reaction with a second
or third hydroperoxide molecule, respectively (Fig. 3A). Because
sulfinic acid and sulfonic acid are not reducible by dihydrolipoamide,
such reactions would result in XfOhr inactivation
and could explain the rate drop at concentrations above 20 μM
LAOOH (Fig. 2A).
We next evaluated the remaining activity of XfOhr after treatment
with several hydroperoxides in the presence of Triton X-100
(Fig. 3B) or Tween-20 (Fig. S4). In the conditions used, t-BHP and
CHP (Fig. 3B, c and d) inactivated XfOhr only slightly. In contrast,
fatty acid hydroperoxides (OAOOH and LAOOH) inactivated
XfOhr at very low concentrations (Fig. 3B, a and b), whereas H2O2
and ChOOH did not affect this peroxidase (Fig. 3B, e and f).
In parallel, the same samples were also analyzed by nonreducing
SDS/PAGE and the pattern of XfOhr inactivation by
various hydroperoxides correlated to its oxidation state (Fig. 3B,
Lower and Fig. S4, Lower). Previously, we have characterized in
detail by mass spectrometry the XfOhr oxidation states corresponding
to each one of the bands (22). The intramolecular
disulfide form of XfOhr has a lower hydrodynamic volume, and
consequently runs faster (band A), than the reduced and overoxidized
forms (band B) of the enzyme (Fig. 3B, Lower and Fig.
S4, Lower). Hyperoxidation (assessed by appearance of band B)
Fig. 2. Lipoamide-lipoamide dehydrogenase–coupled assay for the reduction of lipid hydroperoxides by XfOhr. Peroxidase activity was monitored by the consumption
of NADH at 340 nm in the presence of XfOhr (0.05 μM), lipoamide dehydrogenase from X. fastidiosa (XfLpD, 0.5 μM), and lipoamide (50 μM) in sodium
phosphate buffer (20 mM, pH 7.4) and DTPA (1 mM). (A) Dose-dependent reduction of LAOOH by XfOhr in the presence of Tween-20 (0.25%). (Inset) Plot of the
rates of NADH oxidation, considering points up to 20-μM concentration of LAOOH. (B) Reduction of hydroperoxides (100 μM) in the presence of Triton X-100
(0.15%). PCOOH, phosphatidylcholine hydroperoxide. (C) Peroxidase activity of Ohr on PCOOH, measured after indicated time of treatment with PLA2.
E134 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al.
could only be observed in conditions where some inactivation
was observed (Fig. 3B and Fig. S4).
Inadvertently, Triton X-100 (Fig. 3B, Lower) or Tween-20
(Fig. S4, Lower) provoked oxidation of XfOhr to the intramolecular
disulfide form, even with no addition of hydroperoxide.
This oxidation might be related to the partial structural loss
of XfOhr due to the detergent treatment (Fig. 2A, Inset and Fig.
S2). Another possible explanation is that traces of hydroperoxides
present in detergents could have provoked the oxidation of
XfOhr (35).
For ChOOH, no evidence of a reaction was obtained. These
data agree well with the docking analysis (Fig. 1). However, we
cannot exclude the possibility that ChOOH could not access the
XfOhr active site, because this molecule can form micelles. Indeed,
the parental compound (cholesterol) presents very a low CMC
(25–40 nM) (36). On the other hand, because Triton X-100 and
Tween-20 at least partly solubilize ChOOH (30), our results (Fig.
3B, Lower and Fig. S4, Lower) suggested that this hydroperoxide is
not a good substrate for XfOhr. Also, H2O2 did not inactivate or
hyperoxidize XfOhr in the analyzed conditions (Fig. 3B), consistent
with the low reactivity previously described (22, 23).
For other Cys-based peroxidases, substrate specificity correlates
well with hyperoxidation rates (8, 37–39), which is consistent
with the fact that oxidation to Cys-SOH and hyperoxidation
are both reactions dependent on hydroperoxide concentration
(Fig. 3A). Noteworthy, E. coli Tpx also displays biphasic
Fig. 3. Inactivation/hyperoxidation of Ohr. (A) Schematic catalytic cycle of Ohr and its hyperoxidation: Reaction of reduced Ohr with hydroperoxides generates
the peroxidatic Cys (Cysp)-SOH intermediate, which undergo condensation with resolution Cys (CysR = Cys125). This intramolecular disulfide runs faster than the
reduced form in nonreducing SDS/PAGE (22). The intramolecular disulfide can be reduced back by dihydrolipoamide. In the presence of high amounts of organic
hydroperoxides, Cysp-SOH can be further oxidized to Cysp-SO2H−
(sulfinic acid) or Cysp-SO3H−
(sulfonic acid), which comigrates with the reduced form of Ohr in
SDS/PAGE. Cysp-SO2H−
or Cysp-SO3H−
cannot be reduced by dihydrolipoamide. (B) Oxidative inactivation/hyperoxidation of XfOhr by various hydroperoxides in
the presence of 0.1% Triton X-100. Reduced XfOhr (10 μM) was incubated for 1 h at 37 °C with distinct hydroperoxides at the indicated concentrations: OAOOH
(a), LAOOH (b), t-BHP (c), CHP (d), ChOOH (e), and H2O2 (f). Main panels illustrate residual activity measured by the lipoamide-lipoamide dehydrogenase–coupled
assay, using 200 μM t-BHP as a substrate, in the presence of 0.05 μM XfOhr, 0.5 μM lipoamide dehydrogenase from X. fastidiosa (XfLpD), and 50 μM dihydrolipoamide.
Each point represents an average of triplicates, and the respective bars correspond to the SD. (Insets) Nonreducing SDS/PAGE, with each well corresponding to an
aliquot of Ohr incubated with different concentrations of hydroperoxides (0, 10, 20, 50, and 100 μM). In each well, 2 μg of total protein was applied.
Table 1. Parameters related to hydroperoxide reduction by Ohr
Hydroperoxide
kobs, M−1
·s−1
Oxidative inactivation, μM MICwt/MICΔohr*
AhpE/Ohr competition ∼IC10
†
Extent of inhibition
H2O2 (3.0 ± 0.6) × 103
>100 1
LAOOH (6.3 ± 1.7) × 107
1 >10
OAOOH (4.5 ± 3.5) × 108
1 >10
5(S)-HpETE (2.6 ± 1.2) × 107
ND ND
15(S)-HpETE (6.0 ± 3.0) × 107
ND ND
ChOOH — >100 1
t-BHP 2.0 × 106‡
100 3
ONOOH (2 ± 0.3) × 107
ND —
CHP ND 100 3
kobs, observed rate constant; ND, not determined.
*Ratio of MICwt (minimal concentration of hydroperoxide that inhibits growth of wild-type strain) to MICΔohr
(minimal concentration of hydroperoxide that inhibits growth of Δohr strain).
†
Defined here as the amount of hydroperoxide that provokes 10% Ohr inactivation.
‡
Because Ohr is several orders of magnitude more efficient than MtAhpE in the reduction of t-BHP, it was not
possible to determine this rate constant through the competitive assay. Instead, data from the study by Cussiol
et al. (23) were added here.
Alegria et al. PNAS | Published online December 27, 2016 | E135
BIOCHEMISTRYPNASPLUS
Michaelis–Menten curves with major hyperoxidation by fatty
acid hydroperoxides at concentrations above 10 μM, and H2O2 is
again a poor substrate (37).
Therefore, considering the amount of hydroperoxide that
provokes 10% Ohr inactivation, we estimated that the reactivity
of XfOhr to tested compounds increased in the following order:
H2O2 <<<< CHP < t-BHP < LAOOH ∼ OAOOH (Table 1).
These data are also consistent with the idea that fatty acid hydroperoxides
are the preferred substrates for Ohr proteins.
Competition and Fast Kinetics Assays on the Oxidation of Ohr. Next,
we pursued the determination of the rate constants for the reactions
between XfOhr and various hydroperoxides. This task is
not simple because there are not many assays for organic hydroperoxides
in comparison to the tools available for peroxynitrite
and H2O2. Therefore, we developed a competitive assay
between XfOhr and AhpE from M. tuberculosis (MtAhpE) for
distinct hydroperoxides, monitoring the intrinsic fluorescence of
MtAhpE (further details are provided in Materials and Methods)
and taking advantage of the fact that XfOhr and Ohr from P.
aeruginosa (PaOhr) lack Trp residues, and therefore present no
such intrinsic fluorescence when excited at 295 nm (Fig. S5A).
To validate this AhpE/Ohr competitive assay, we first investigated
t-BHP, because the corresponding rate constants are already
known for both Cys-based peroxidases. For instance, the rate
constant for the reaction between t-BHP and MtAhpE is quite low,
8 × 103
M−1
·s−1
(8), compared with the corresponding value for
XfOhr and t-BHP, which is two to three orders of magnitude higher
(23). As expected, XfOhr strongly inhibited the drop in MtAhpE
fluorescence induced by t-BHP (Fig. 4A).
We went on to evaluate the competition between XfOhr and
MtAhpE for several organic hydroperoxides. As an illustrative
example, we show here the competition of the two Cys-based
peroxidases for 15-hydroperoxyeicosatetraenoic acid [15(S)HpETE],
an arachidonic acid hydroperoxide. Reduced XfOhr
inhibited the decrease of MtAhpE fluorescence caused by 15(S)HpETE
in a concentration-dependent manner (Fig. 4B), and the
second-order rate constant between XfOhr and 15(S)-HpETE
was determined to be 6 ± 3 × 107
M−1
·s−1
. As anticipated, XfOhr
previously alkylated with N-ethylmaleimide (NEM) completely
lost its ability to inhibit MtAhpE oxidation (Fig. S5B).
A second example is the competition between the two peroxidases
for H2O2, considering that the rate constant of MtAhpE for
H2O2 is 8.1 × 104
M−1
·s−1
(8). We observed that only at very high
concentrations (10–30 μM) was XfOhr able to inhibit MtAhpE
(2 μM) oxidation (Fig. 4C). The rate constant of the reaction
between XfOhr and H2O2 was estimated to be 3.0 ± 0.6 × 103
M−1
·s−1
.
Once again, XfOhr blocked with NEM lost its capacity to compete
with MtAhpE for H2O2. Previously, by a steady-state approach (the
lipoamide-lipoamide dehydrogenase–coupled assay), we determined
that the catalytic efficiency (kcat/Km) of XfOhr for H2O2 was (2.3 ±
0.2) × 102
M−1
·s−1
(23).
This Ohr/AhpE competition assay was performed for several
hydroperoxides of distinct chemical structures (Fig. S6), and it was
possible to establish the respective rate constants (Table 1). PaOhr
was also assessed using this competitive assay, and essentially the
same results were obtained as for the enzyme from X. fastidiosa.
For instance, the second-order rate constant between OAOOH
and PaOhr is also in the range of 108
M−1
·s−1
(Fig. S6J).
Once again, the same trend was observed: Ohr proteins displayed
higher reactivity toward hydroperoxides with an elongated
shape and hydrophobic properties. However, it was not
possible to determine the rate constant for the reaction between
XfOhr and ChOOH because the intrinsic fluorescence of
MtAhpE did not change upon treatment with this oxidant. One
exception was peroxynitrite, which is described below.
Reactivity of XfOhr Toward Peroxynitrite. Pathogens can be exposed
to peroxynitrite during host–pathogen interactions (40–42). Because
the reactivity of Ohr proteins toward peroxynitrite has not been
hitherto reported, this analysis was performed here. Reduced XfOhr
caused peroxynitrite to decay faster, in a dose-dependent manner
(Fig. 5A). The reaction was so rapid that it precluded the determination
of the rate constant by this direct approach. Rather, the rate
constant was determined by two independent competitive assays
against HRP (11) or against MtAhpE (as described above). XfOhr at
concentrations considerably lower than HRP (10 μM) inhibited the
formation of compound I by peroxynitrite (0.4 μM) (Fig. 5B). From
the dose–response effect of XfOhr on peroxynitrite-mediated HRP
compound I formation, an apparent rate constant of (1.2 ± 0.4) ×
107
M−1
·s−1
at pH 7.4 and 25 °C was determined (Fig. 5B, Inset).
As an alternative, we also analyzed the competition between
MtAhpE and XfOhr for peroxynitrite. Peroxynitrite (0.5 μM) caused
a rapid decrease in total fluorescence intensity (λex = 295 nm) of
reduced MtAhpE (2.5 μM). The exponential fitting of the experimental
curve was consistent with a rate constant of peroxynitritemediated
MtAhpE oxidation of 1.1 × 107
M−1
·s−1
at pH 7.4 and
25 °C, in close agreement to the previously reported value (8). Reduced
XfOhr dose-dependently inhibited the decrease in fluorescence
of MtAhpE caused by peroxynitrite, consistent with a rate
constant between peroxynitrite and XfOhr of (2.0 ± 0.3) ≥ 107
M−1
·s−1
at pH 7.4 and 25 °C (Fig. 5C). Therefore, XfOhr reduces not only
Fig. 4. AhpE/XfOhr competitive assay for distinct hydroperoxides. Reactions were carried out in potassium phosphate buffer (100 mM) and DTPA (50 μM),
pH 7.4, at 25 °C. (A) MtAhpE (2 μM) and t-BHP (1.8 μM) in the presence (red line) or absence (black line) of XfOhr (2 μM). (B) Emission spectrum of MtAhpE
(2 μM) in the reduced state (black) and after oxidation by 1.7 μM 15(S)-HpETE (red). To the reaction mixture represented by the red line, XfOhr was added at
the following concentrations: 1.0 μM (brown), 2.0 μM (blue), 4.0 μM (green), and 8.0 μM (purple). (C) Emission spectrum of AhpE (2 μM) in the reduced state
(black) and after oxidation by 1.8 μM hydrogen peroxide (red). To the reaction mixture represented by red line, XfOhr was added at the following concentrations:
10.0 μM (blue) and 30.0 μM (green). RFU, relative fluorescence units.
E136 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al.
fatty acid hydroperoxides but also peroxynitrite, with extraordinarily
high rates.
In Vivo Characterization of Ohr Sensitivities Toward Various Hydroperoxides.
We aimed to investigate if these results on substrate specificity
correlate to bacterial responses toward distinct hydroperoxides.
For this purpose, we used P. aeruginosa, which produces surfactants
and secretes lipases, allowing it to solubilize and take up very hydrophobic
compounds (43).
The sensitivities of Δohr and wild-type strains were comparatively
analyzed by the minimal inhibitory concentration (MIC) assay
against various hydroperoxides. Interestingly, none of the lipid hydroperoxides
in the concentration range studied here (OAOOH,
LAOOH, and ChOOH) inhibited the growth of the wild-type strain,
whereas H2O2, t-BHP, and CHP did (Fig. 6). As expected, the Δohr
strain presented a reduced capacity to grow in the presence of either
artificial (CHP and t-BHP) or fatty acid hydroperoxides compared
with the wild type (Fig. 6). It is noteworthy that the extent of inhibition
(defined here as MICwild-type strain/MICΔohr strain) for
OAOOH and LAOOH (more than 10-fold) was considerably
greater than a similar parameter for artificial hydroperoxides
(around threefold for t-BHP and CHP). For H2O2, the MIC was the
same for the wild-type and Δohr strains (Table 1) and ChOOH did
not inhibit the growth of either. These results indicated once again
that fatty acid hydroperoxides are the best substrates for Ohr proteins,
which correlates well with the original docking analysis (Fig. 1
and Fig. S1), the inactivation assay data (Fig. 3B and Fig. S4), and
the second-order rate constant (k) values (Table 1). As important
controls, the phenotypes of the Δohr strain were complemented in
trans (pUCp18 plasmid) with the ohr gene from P. aeruginosa
(LN04 strain) with the restoration of the wild-type resistance to
hydroperoxides (Fig. 6). These controls unequivocally indicated that
the observed sensitivities were due to the ohr gene deletion. Furthermore,
nonoxidized lipids did not kill either wild-type or Δohr
strains (Fig. S7 A–C), indicating that the hydroperoxide moiety in
the carbon backbone was fundamental for inducing cell death.
The response of P. aeruginosa to fatty acid hydroperoxides was
further investigated by analyzing other mutants to thioldependent
peroxidases. Remarkably, among all mutants, only Δohr
displayed sensitivity to LAOOH (Fig. 7 A and B), indicating the
PaOhr is essential to the protection of P. aeruginosa to this insult.
Furthermore, the Δohr strain transformed with genes for other
thiol-dependent peroxidases did not recover the wild-type phenotype.
For example, Gpx expression in the Δohr background
only partially restored the resistance to LAOOH treatment
(Fig. 7A). Expression of thiol-dependent peroxidases in the Δohr
background was ascertained by Western blot (Fig. S8A). Finally,
as expected for a gene central to the bacterial response to fatty
acid hydroperoxides, treatment with LAOOH led to higher levels
of ohr induction than did t-BHP (Fig. 7C and Fig. S8B). For
comparison purposes, we also show here that the expression of
the ohr gene in the ΔohrR background was maximal, because the
repression by the transcription factor OhrR was absent (Fig. 7C).
In contrast to the LAOOH insult, the response of P. aeruginosa
to exogenous H2O2 was highly dependent on catalase, because
increased sensitivity toward the wild-type strain was only
detected in cells treated with 3-amino-1,2,4-triazole (ATZ), a
Fig. 5. Peroxynitrite reduction by XfOhr. Reactions were carried out in potassium phosphate buffer (100 mM), DTPA (100 μM), pH 7.4, at 25 °C. (A) Peroxynitrite
(50 μM) was rapidly mixed with reduced Ohr at increasing concentrations, and oxidant decay was followed by its intrinsic absorbance at 310 nm. (B) Peroxynitrite
(0.4 μM) was mixed with HRP (10 μM) in the absence and presence of increasing concentrations of reduced Ohr. HRP-compound I formation was monitored at the
Soret band. The line represents the expected results simulated using the Gepasi program (76), and considering a simple competition between reduced XfOhr and
HRP for peroxynitrite, a rate constant of HRP-mediated peroxynitrite reduction of 2.2 × 106
M−1
·s−1
and a rate constant of XfOhr-mediated peroxynitrite reduction
of 1.2 × 107
M−1
·s−1
, calculated according to Eq. 1. (Inset) Time course of compound I formation by the reaction of HRP (10 μM) with peroxynitrite (0.4 μM) in the
absence or presence of increasing concentrations of reduced Ohr. (C) Peroxynitrite (0.5 μM) was mixed with reduced MtAhpE (2.5 μM) in the absence or presence of
increasing concentrations of reduced XfOhr, and MtAhpE oxidation was followed by its total intrinsic fluorescence decrease (λ = 295 nm).
Fig. 6. Susceptibility of Δohr P. aeruginosa strain to various hydroperoxides.
The MIC assay was performed to assess the sensitivities of P. aeruginosa strains
to distinct hydroperoxides: pUCp18 Ø is an empty vector, and pUCp18 ohr is a
vector for expression of ohr from P. aeruginosa. The concentrations of hydroperoxides
used are described above each panel. Plates were incubated at 37 °C
under agitation (200 rpm), and the results were observed after 16 h. MIC assays
were done in biological triplicates, each one with two technical replicates. All
hydroperoxides were dissolved in DMSO, with the exception of ChOOH, which
was dissolved in isopropyl alcohol. As control experiments, the same assays were
performed with 5% DMSO or 5% isopropyl alcohol only or with the respective
fatty acid or cholesterol dissolved in DMSO and isopropyl alcohol, respectively,
and no loss of viability were observed. Wt, wild type.
Alegria et al. PNAS | Published online December 27, 2016 | E137
BIOCHEMISTRYPNASPLUS
catalase inhibitor (Fig. S7 D–F). This observation is in agreement
with studies in E. coli, P. aeruginosa, and Francisella tularensis,
where catalase enzymes are preponderant in the defense against
exogenous H2O2 (44–47).
The response of P. aeruginosa to peroxynitrite is more difficult
to investigate, in part, due to the intrinsic instability of this
hydroperoxide. In this case, we had to use a distinct assay, different
from MIC, by exposing the bacterium to 3-morpholinosydnonimine
hydrochloride (SIN-1, a peroxynitrite generator) in buffer and then
assessing the number of viable colonies. A tendency of an increased
sensitivity for Δohr cells in relation to the wild-type strain was detected
(Fig. S8D), which was statistically significant in cells, where
catalase was inhibited by ATZ (Fig. 7D). In contrast, both ATZtreated
and untreated wild-type cells were equally sensitive to
SIN-1, and deletion of ahpC gene did not affect the viability of
bacterial cells (Fig. S8C). Furthermore, the expression of ohr was
induced in cells exposed to SIN-1 (Fig. 7C), implicating PaOhr in
the response of P. aeruginosa to peroxynitrite.
Discussion
Previous work has indicated that substrate hydrophobicity is a
major physicochemical determinant of substrate specificity for
Ohr (16, 19, 21, 22). Accordingly, docking analysis performed here
with XfOhr indicated that nonpolar contacts appear to be major
determinants in the binding of lipid hydroperoxides within the
active site (Fig. 1 and Fig. S1A). The elongated shape of the substrate
appears to be another feature to be considered presumably
because the substrates, once docked, fill a Y-shaped cavity on the
enzyme generating a large hydrophobic contact area, which would
be anticipated to contribute significantly to the interaction energy.
Indeed, cholesterol, which is highly hydrophobic but with a shape
very distinct from PEG and fatty acids, did not dock well into the
XfOhr active site (Fig. S1B). Other biochemical (Figs. 2 and 3) and
microbiological (Fig. 6) assays also indicated that ChOOH is not an
Ohr substrate. For example, in the case of the MIC assay (Fig. 6),
in addition to the natural surfactants of P. aeruginosa, Brij-58 (1%)
was supplemented during the experiments. Despite this fact, the
Δohr strain was no more sensitive to ChOOH than the wild-type
strain, indicating a lack of significant affinity between ChOOH and
the enzyme.
The AhpE/Ohr competitive assay developed here was straightforward
in comparison to other methods previously described,
allowing us to obtain the second-order rate constants for the oxidation
of peroxidatic Cys by distinct hydroperoxides. Importantly,
we could use fatty acid hydroperoxides in the low micromolar
range (1–2 μM), which is well below the CMC of long-chain fatty
acid hydroperoxides (10–40 μM). With this approach (associated
with the inactivation and the MIC assays), we could unambiguously
establish that long-chain fatty acid hydroperoxides are more efficiently
reduced by Ohr than artificial organic hydroperoxides and
H2O2 (Table 1).
In addition to fatty acid hydroperoxides, peroxynitrite was reduced
with very high efficiency by XfOhr (on the order of 107
M−1
·s−1
).
This finding was somehow unexpected because peroxynitrite is not a
hydrophobic molecule, although the reaction of peroxynitrite with
thiols involves the protonated form of peroxynitrite (peroxynitrous
acid) and thiolates (48). It should be noted here that reactions
of peroxynitrite with thiolates are much faster than similar reactions
of thiolates with organic hydroperoxides or H2O2 (48–50). Unfortunately,
there is no report of the kinetics between fatty acid
hydroperoxides with noncatalytic thiols, such as free Cys or the single
reduced Cys residue in BSA. Nevertheless, using the reaction between
t-BHP and ordinary thiolates as a reference (51), it is evident
that the gain in reactivity afforded by Ohr is two to three orders of
magnitude for H2O2 and peroxynitrite, but six orders of magnitude
for t-BHP (Table S1).
According to the Brønsted relationship, the pKa of the leaving
group is a factor that is involved in the reactivity of thiolates with
hydroperoxides (13). The pKa values for aliphatic alkoxyl groups,
which are the leaving groups corresponding to t-BHP and fatty
acid hydroperoxides, are expected to have similar values (13).
Therefore, it is reasonable to think that the reactions of t-BHP
and fatty acid hydroperoxides with ordinary thiols (e.g., the thiol
of BSA) are within the same range and that the catalytic power
of Ohr proteins for fatty acid hydroperoxides is therefore extraordinary
(at least 106
-fold increase). This high reactivity is
probably related to its capacity to bind and orient the peroxyl
group of fatty acid hydroperoxides toward the thiolate group of
Cys61
(Fig. 1), decreasing the free energy of the transition state.
In the case of the reduction of peroxynitrite by Ohr, the corresponding
rate constant is one of the highest determined so far
(52). In fact, the rate constant of peroxynitrite reduction by Ohr
is comparable to the rate constants reported for Prxs and thiolor
selenol-dependent Gpxs (10, 12, 13, 53–55). Therefore, it is
feasible that Ohr plays a relevant role in vivo in the reduction of
peroxynitrite. This role might be significant, especially considering
that inducible nitric oxide synthase (and possibly peroxynitrite
as a product of nitric oxide) displays a crucial role in the
pulmonary response to P. aeruginosa infection (42, 56). Also
for plant pathogens (e.g., X. fastidiosa), peroxynitrite and other
nitric oxide-mediated processes are emerging as relevant players
in defense responses (41). Accordingly, the levels of PaOhr are
induced in cells exposed to the peroxynitrite generator (Fig. 7C),
and the Δohr strain treated with ATZ is sensitive to this oxidant
(Fig. 7D). However, at the moment, it is difficult to identify if the
response of P. aeruginosa to SIN-1 is a direct effect of peroxynitrite
or if it is due to some secondary derived oxidant. For instance, it is
well known that lipids are targets for peroxynitrite oxidation and
could release secondary products that might mount the adaptive
response of P. aeruginosa (57). The involvement of catalase in
the response of bacteria to peroxynitrite was shown previously in
F. tularensis and Salmonella enterica serovar Typhimurium, two microorganisms
devoid of Ohr (47, 58), although the latter does
contain an osmC gene. Our data indicate that especially Ohr and
Fig. 7. Response of P. aeruginosa to LAOOH and peroxynitrite. pUCp18 Ø is an
empty vector, and pUCp18 ohr, ahpC1, tpx, and gpx from P. aeruginosa are
vectors for ohr, ahpC1, tpx, and gpx expression, respectively. (A and B) MIC
assay to assess the sensitivities of P. aeruginosa strains deficient for Cys-based
peroxidases to LAOOH. (C, Upper) Western blot for Ohr expression in P. aeruginosa
using an antibody against Ohr from X. fastidiosa. (C, Lower) Ponceau S
was used as a loading control. Cells were exposed to SIN-1 (1 mM), DMSO (5%),
LAOOH (50 μM), t-BHP (200 μM), and MeOH (5%) for 30 min at 37 °C. WT, wild
type. (D) Colony count assay to assess the sensitivities of P. aeruginosa strains to
SIN-1 (3 mM). In all cases, cells were treated with ATZ (5 mM), a catalase inhibitor,
10 min before cells were challenged with SIN-1 (3 mM) for 30 min at
37 °C. The bars represent the means of the colony formation unit (c.f.u.) percentage
relative to the sensitivity of cells treated with DMSO (5%) + 3-ATZ
(5 mM) plus SD. The Δohr mutant was statistically more sensitive than the wildtype
strain (**P < 0.05, unpaired t test; n = 8). WT, wild type.
E138 | www.pnas.org/cgi/doi/10.1073/pnas.1619659114 Alegria et al.
possibly catalase cooperate in the protection of P. aeruginosa
against this oxidant (Fig. 7).
In addition to peroxynitrite, fatty acid hydroperoxides are involved
in host–pathogen interactions (59, 60). Fatty acid hydroperoxides
could be generated by nonenzymatic lipid peroxidation
of cis-vaccenic acid and other monounsaturated fatty acids that
are present in bacterial membranes, because most bacteria synthesize
little or no polyunsaturated fatty acids (61). For this
reason, it is assumed that nonenzymatic lipid peroxidation is not
a favorable process in bacteria. Alternatively, Ohr may reduce
polyunsaturated fatty acid hydroperoxides enzymatically generated
by plant or animal hosts. In mammals, pathogens, such as
P. aeruginosa, can activate host cytosolic PLA2 (62), releasing
arachidonic acid (20:4 ratio) for oxidative modification by enzymes,
such as 5-lipoxygenase or 15-lipoxygenase (63). Products
of lipoxygenases have profound and distinct effects on the outcome
of inflammation (59, 64). Given that endogenous levels of
fatty acid hydroperoxides, such as 5-hydroperoxyeicosatetraenoic
acid [5(S)-HpETE] or 15(S)-HpETE, regulate acute inflammation
(59), it is reasonable to hypothesize that Ohr might somehow impact
the progression of the immune response. In plants, the immune
response is also mediated by oxidized products of fatty acids,
derived from linoleic (18:2 ratio) and linolenic (18:3 ratio) acids in
this case (60). Therefore, it is tempting to speculate that Ohr might
subvert host immune pathways by some still unknown mechanisms
(65). Remarkably, P. aeruginosa contains endogenous enzymatic
systems capable of oxidizing unsaturated fatty acids (especially
oleic acid) to hydroperoxide and hydroxide derivatives, whose biological
function is still elusive (66, 67). Moreover, the experimental
approach presented herein allowed dissecting the relevance
of Ohr toward three different groups of biologically relevant hydroperoxides,
underscoring the complementary role that different
thiol- and heme-dependent peroxidases have on controlling cellular
oxidative signaling and damage.
Materials and Methods
Reagents, Including Hydroperoxides. All reagents purchased had the highest
degree of purity. The t-BHP was obtained from Sigma–Aldrich Co. CHP and
H2O2 were obtained Merck KGaA. XfOhr and PaOhr, as well as MtAhpE,
were obtained and, in some experiments, reduced as previously described (8,
55). The 15(S)-HpETE and 5(S)-HpETE were purchased from Cayman Chemical.
Photochemical Synthesis of Lipid Hydroperoxides. Oleic acid, linoleic acid,
cholesterol, and phosphatidylcholine were photooxidized to the corresponding
hydroperoxides according to the method of Miyamoto et al. (68).
This procedure is based on the oxidation of lipids by singlet molecular oxygen
produced in an O2-saturated atmosphere using methylene blue as a
photosensitizer. OAOOHs, LAOOHs, and ChOOHs were purified by flashcolumn
chromatography and were quantified by iodometry (68). The hydroperoxide
isomers were not separated among themselves, only from the
parental lipids. Phosphatidylcholine hydroperoxide was purified by HPLC
and was quantified by phosphorus assay and iodometry (69).
Docking Lipid Hydroperoxides in Ohr. Using the X. fastidiosa Ohr structure
(PDB ID code 1ZB8) as a template, docking analysis was carried out using
Gold software, version 5.0 (70), utilizing organic hydroperoxides with distinct
chemical structures.
Lipoamide-Lipoamide Dehydrogenase Peroxidase-Coupled Assay. The lipoyl
peroxidase activity of Ohr was determined as previously described (23). Reactions
were followed by decay of A340 (e = 6,290 M−1
·cm−1
) due to NADH
oxidation. In some cases, several hydrophobic hydroperoxides were solubilized
in Tween-20 or Triton X-100 as described throughout the main text and
appropriate controls were performed.
Mass Spectrometry Analysis of the Products Formed in the Ohr Reaction with
LAOOH and OAOOH. OAOOH or LAOOH (4 μM) was incubated in the presence
of an equimolar concentration of reduced XfOhr for 1 min at 37 °C in sodium
phosphate buffer (20 mM), pH 7.4, containing diethylenetriaminepentaacetic
acid (DTPA) (0.1 mM). As a control reaction, OAOOH and LAOOH were
incubated with previously alkylated Ohr (0.2 mM NEM). Afterward, an organic
extraction was performed, and the products were analyzed by HPLCtandem
mass spectrometry. Mass spectrometry analysis was performed in a
triple-quadrupole instrument (Quattro II; Micromass) essentially as described
by Miyamoto et al. (68).
Analysis of Ohr Hyperoxidation and Inactivation. Freshly purified recombinant
XfOhr (300 μM) was reduced for 1 h using DTT (100 mM). Afterward, excessive
DTT was removed by gel filtration (PD-10 desalting; GE Healthcare), and the
amount of protein recovered was quantified by its A280 (e = 3,960 M−1
·cm−1
;
determined by the ProtParam tool ExPASy). Next, XfOhr (10 μM) was treated
with increasing amounts of hydroperoxides (as depicted in Fig. 3B and Fig. S4)
for 1 h at 37 °C in sodium phosphate buffer (20 mM), pH 7.4, containing DTPA
(1 mM) and 0.15% (vol/vol) Triton X-100 or 0.25% (vol/vol) Tween-20.
After these pretreatments, XfOhr was again submitted to gel filtration to
remove excessive hydroperoxides, and the recovered enzyme was again
quantified by A280. Finally, the same sample of XfOhr was assayed by the
lipoamide-lipoamide dehydrogenase–coupled assay (using t-BHP as a substrate)
and by nonreducing SDS/PAGE, which allow the assessment of the
XfOhr oxidative state (22).
Digestion of Phosphatidylcholine Hydroperoxide with PLA2. This enzymatic
digestion was performed as previously described (71). In a solution containing
CaCl2 (10 mM), KCl (35 mM), and Tris (20 mM) at pH 8.0, phosphatidylcholine
(catalog no. P3556; Sigma) from egg yolk was added at a final
concentration equal to 100 μM. The solution was stirred for 1 min abruptly.
Thereafter, PLA2 (P0861 Sigma–Aldrich Co.) was added (final amount of
10 units), and the solution was then incubated for 15 min or 30 min at 40 °C
under gentle agitation. After this time, the components of the dihydrolipoamide
system and Triton X-100 were added for testing the activity of XfOhr on
phospholipids treated with PLA2.
Kinetics of Organic Hydroperoxide Reduction by Ohr. A competition method
was developed here, taking advantage of the fact that Ohr from X. fastidiosa
and P. aeruginosa does not possess Trp residues, and consequently
displays low intrinsic fluorescence. In contrast, MtAhpE has intrinsic fluorescence
that decreases upon oxidation (8, 55). As expected, XfOhr practically
lacks intrinsic fluorescence when excited at 295 nm, in contrast to
MtAhpE (Fig. S5). Therefore, the assay developed here is based on the
competition between Ohr and MtAhpE for distinct hydroperoxides, monitoring
the intrinsic fluorescence of MtAhpE when exciting at 295 nm, either
following the time course of the reaction (using an SX20 Applied Photophysics
stopped-flow apparatus) or at final time points (using an Aminco
Bowman Series 2 luminescence spectrophotometer or a Cary Eclipse Fluorimeter).
Rate constants for MtAhpE oxidation were determined for those
organic hydroperoxides that were not previously studied by Reyes et al. (8)
and Hugo et al. (55).
Concentrations of MtAhpE, Ohr, and hydroperoxide used were typically
2 μM, 2–8 μM, and 1.8 μM, respectively. Because enzyme concentration decreased
during the time course of the experiment, no pseudo–first-order
conditions applied. Therefore, we used the following equation (Eq. 1) to find
out the relationship between the rate constants for the reaction of a selected
hydroperoxide with Ohr (kOHR) and with MtAhpE (kAhpE):
kC.  Prot
kOHR
=
ln
n
½red
C.   ProtŠ0
½red
C.   ProtŠ0 − ½oxC.   ProtŠ∞
o
ln
n ½red
OHRŠ0
½red
OHRŠ0 − ½oxOHRŠ∞
o , [1]
where kC. Prot is the rate constant for the reaction between a selected hydroperoxide
with the protein competing with Ohr for it (in this case, reduced
MtAhpE, whose reactivity with the hydroperoxide is either already known or
determined herein, as described in Fig. S6); kOHR is the rate constant of the
reaction between the hydroperoxide with reduced Ohr; [redC. Prot]0 is the initial
concentration of the reduced competing protein; [oxC. Prot]∞ corresponds to
the final concentration of the competing protein in the oxidized state; [redOhr]0
is the initial concentration of reduced Ohr; and [oxOhr]∞ is the final concentration
of oxidized Ohr, determined as [oxC. Prot]∞ formed in the absence minus
the presence of Ohr (50). The final concentration of MtAhpE in the oxidized
state was determined assuming an equimolar reaction with hydroperoxide in
the absence of the competing protein.
Kinetics of Peroxynitrite Reduction by Ohr. The reduction of peroxynitrite by
XfOhr was investigated using different methodologies. First, the initial rate
of peroxynitrite decomposition was followed at 310 nm due to its intrinsic
absorbance in the absence and presence of increasing concentrations of
reduced XfOhr in 100 mM (pH 7.4) sodium phosphate buffer containing
Alegria et al. PNAS | Published online December 27, 2016 | E139
BIOCHEMISTRYPNASPLUS
DTPA (0.1 mM) at 25 °C, using an SX20 Applied Photophysics stopped-flow
apparatus (50). In a second approach, the kinetics of peroxynitrite reduction
were indirectly determined using a competition assay between reduced XfOhr
and HRP for peroxynitrite (10, 11, 50). HRP compound I formation was measured
at various concentrations of reduced XfOhr, and experimental curves
were fitted to exponential curves before the slow decay of compounds I –II
occurred. Because no pseudo–first-order conditions applied (reduced XfOhr
concentration decreased during the time course of the experiment), we used
Eq. 1 to find out the relationship between the kHRP (the C. Prot in this assay)
and kOHR.
The final concentration of compound I was quantitated using the reported
e398 nm = 42,000 M−1
·cm−1
and the final concentration of oxidized XfOhr,
determined as compound I formed in the absence minus the presence of
XfOhr (11). To perform these calculations, we previously determined the
rate constant between HRP and peroxynitrite as 2.2 × 106
M−1
·s−1
at pH 7.4
and 25 °C for the specific batch and experimental conditions used in the
experiments described here. Indeed, the rate constant changed from batch
to batch of HRP (72).
Finally, the competition approach between reduced Ohr and MtAhpE
described above was also used for determining the kinetics of peroxynitritemediated
MtAhpE oxidation.
Strains and Growth Conditions. P. aeruginosa UCBPP-PA14 (73) wild-type and
MAR2xT7 mutants (74) (see Table S2), and E. coli strains were grown in
lysogenic broth (LB) medium at 37 °C supplemented, when necessary, with
gentamicin (10 μg/mL), carbenicillin (300 μg/mL), or ampicillin (100 μg/mL).
Cloning for Complementation Assays in P. aeruginosa. The coding region from ohr
gene (PA14_27220) with its 150-bp upstream sequence and ahpC (PA14_01710),
ahpC2 (PA14_18690), tpx (PA14_31810), and gpx (PA14_27520) genes (corresponding
to fragments of 780 bp, 561 bp, 600 bp, 485 bp, and 490 bp, respectively)
were amplified using oligonucleotides described in Table S3). With
the exception of ohr, all genes were cloned without a stop codon and harboring
a human influenza hemagglutinin (HA) tag inserted at 3′, allowing us to
check their expression levels in P. aeruginosa cells by Western blotting.
The final products were cloned into EcoRI and BamHI sites of the broadhost
range vector pUCp18 (GenBank accession no. U07164) previously
digested with the same enzymes. The pUCp18 constructs were introduced
into P. aeruginosa PA14 wild-type and MAR2xT7 transposon ohr mutant
(Δohr) cells by electroporation (75). As a control, the same strains were
transformed with empty pUCp18 vector. All PCR reactions were carried out
with Thermo Scientific High Fidelity Taq Polymerase using genomic DNA of
PA14 as a template. Errors from PCR reactions were checked by sequencing
the clones.
MIC Assay. Cultures of P. aeruginosa (PA14 and Δohr) were grown on LB
medium at 37 °C until OD600 = 1. After that time, cultures were diluted in
fresh LB medium supplemented with an appropriate antibiotic to OD600 =
0.01; then, 150 μL was dispensed in 96-well plates previously loaded with
different concentrations of tested hydroperoxides. Afterward, assay plates
were incubated at 37 °C under agitation (200 rpm), and the results were
recorded after 16 h. To increase the solubility of ChOOH, LB medium was
supplemented with 1% Brij-58. Concentrations of hydroperoxides and conditions
of solubilization are described in Figs. 6 and 7 and Fig. S7.
Colony Counting Assay to Assess SIN-1 Sensitivity. Cultures of P. aeruginosa
were grown on LB medium at 37 °C until OD600 = 1. After that point,
cultures were washed twice in Dulbecco PBS (Life Technologies) and diluted
to OD600 = 0.01 in the same buffer. A volume of 200 μL of the cell
suspension was incubated with 5 mM catalase inhibitor ATZ for 10 min at
room temperature without agitation. After that time, 6.2 μL of 96 mM
SIN-1 solution (diluted in DMSO) was added at the bottom of the tube and
incubated for 30 min at 37 °C at 200 rpm, and treated cultures were then
serially diluted in 10 mM MgSO4 and plated on solid LB medium to count
the number of viable cells by colony formation unit. As a control, the same
treatment was carried out only with DMSO. The results were expressed by
the relative percentage, compared with controls, of viable cells remaining
after treatment. The statistical analysis was performed using an unpaired
t test (P < 0.05).
Ohr Expression upon Oxidative Insults. Cultures of P. aeruginosa (PA14, Δohr,
and ΔohrR) were grown on M9 medium supplemented with 0.2% glucose at
37 °C and 200 rpm until OD600 = 0.5. After that time, 500 μL of each culture
was separated in different flasks to receive the treatments (200 μM t-BHP,
50 μM LAOOH, and 1 mM SIN-1). As a control, the same volumes of the
solvents in which the previous compounds were diluted [water, 5% (vol/vol)
methanol, and 5% (vol/vol) DMSO, respectively] were added. After 30 min of
treatment (37 °C, 200 rpm), cells were pelleted and then resuspended in
Laemmli buffer and processed for SDS/PAGE analysis. The gel was electroblotted
onto a 0.45-μm nitrocellulose membrane in Tris·glycine buffer at 24
V/600 mA for 30 min. Blotted membranes were blocked for 3 h at room
temperature in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T)
and 5% (vol/wt) nonfat dry milk (Bio-Rad). The membrane was then incubated
with a rabbit anti-XfOhr polyclonal serum (1:1,000 in TBS-T) overnight
at 4 °C, followed by washing with TBS-T. The membrane was
subsequently incubated with an anti-rabbit IgG-alkaline phosphatase antibody
(KPL, Inc.; 1:1,000 in TBS-T) for 2 h. After washing, the proteins were
detected following incubation with alkaline phosphatase substrates: 5-bromo-
4-chloro-3-indolyl phosphate (0.3 mg/mL)/nitro blue tetrazolium (0.1 mg/mL)
on an appropriate buffer [10 mM Tris·HCl (pH 9.0), 100 mM NaCl, and
5 mM MgCl2].
ACKNOWLEDGMENTS. We thank Dr. Regina Baldini [Instituto Quimica, Universidade
de São Paulo (USP)] and Laurence G. Rahme (Harvard University) for
providing PA14 and strains from a transposon library. We also thank Adriano de
Britto Chaves-Filho (Instituto Quimica, USP) for his support in mass spectrometry
analysis. This work was financially supported by Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) Grant 13/07937-8 (Redox Processes
in Biomedicine) (to M.A.d.O., S.M., P.D.M., O.A., and L.E.S.N.), NIH Grant
1R01AI095173 (to R.R.), a grant from the Universidad de la República (Comisión
Sectorial de Investigación Científica, Uruguay; to R.R.), and a grant from
Ridaline (Fundación Manuel Pérez) (to R.R.). M.H. was the recipient of a fellowship
from the Agencia Nacional de Investigación e Innovación (Uruguay), and T.G.P.A.
(2008/07971-3) and D.A.M. (2012/21722-1) were recipients of fellowships from
FAPESP.
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