Báo cáo Y học: Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities pptx

Two GPX-like proteins from
Lycopersicon esculentum
and
Helianthus
annuus
are antioxidant enzymes with phospholipid hydroperoxide
glutathione peroxidase and thioredoxin peroxidase activities
Ste
´
phane Herbette
1
, Catherine Lenne
1
, Nathalie Leblanc
1
, Jean-Louis Julien
1
, Joe¨ l R. Drevet
2
and Patricia Roeckel-Drevet
1
1
UMR 547-PIAF INRA/Universite
´
Blaise Pascal, Aubie
`
re, France;
2
UMR 6547-GEEM CNRS, Universite
´
Blaise Pascal,

Laboratoire Epididyme & Maturation des Game
`
tes, Aubie
`
re, France
This study investigated the enzymatic function of two
putative plant GPXs, GPXle1 from Lycopersicon esculentum
and GPXha2 from Helianthus annuus, which show sequence
identities with the mammalian phospholipid hydroperoxide
glutathione peroxidase (PHGPX). Both purified recombin-
ant proteins expressed in Escherichia coli show PHGPX
activity by reducing alkyl, fatty acid and phospholipid
hydroperoxides but not hydrogen peroxide in the presence
of glutathione. Interestingly, both recombinant GPXle1
and GPXha2 proteins also reduce alkyl, fatty acid and
phospholipid hydroperoxides as well as hydrogen peroxide
using thioredoxin as reducing substrate. Moreover, thio-
redoxin peroxidase (TPX) activities were found to be higher
than PHGPX activities in terms of efficiency and substrate
affinities, as revealed by their respective V
max
and K
m
values.
We therefore conclude that these two plant GPX-like pro-
teins are antioxidant enzymes showing PHGPX and TPX
activities.
Keywords: antioxidant; free radical scavenger; tomato; sun-
flower.
In all aerobic organisms, reactive oxygen species (ROS)

originating from the metabolism of oxygen constitute a
threat to virtually any cell constituent. In plants, it has
been shown that environmental stresses can cause an
increase in ROS levels [1–4]. Despite their noxious effects
on proteins, lipids and nucleic acids, which could ulti-
mately lead to cell death, ROS, in a more controlled
manner, can participate in early signaling pathways in
responses to both biotic and abiotic stresses [5,6]. To cope
with elevated levels of ROS, plants have evolved different
enzymatic and nonenzymatic mechanisms. In the latter are
found reducing molecules such as carotene, tocopherol,
ascorbate, Fe
2+
, glutathione, while the antioxidant enzy-
matic equipment is composed of several enzymes including
superoxide dismutase (SOD), ascorbate peroxidase (APX),
catalase, glutathione reductase (GR), glutathione peroxi-
dase (GPX), glutathione-S-transferase (GST) or thiore-
doxin peroxidase (TPX).
In mammals, the GPX family of proteins can be divided
into five clades according to their amino-acid sequence,
substrate specificity and subcellular localization; the cyto-
solic GPX (GPX1), the gastro-intestinal GPX (GPX2), the
plasma GPX (GPX3), the phospholipid hydroperoxide
GPX (GPX4) and selenoindependent epididymis GPX
(GPX5) [7,8].
To date, cDNAs encoding proteins similar to animal
GPX have been isolated from different plants and have been
shown to be induced by biotic and abiotic stresses [9–11].
Plant GPX-like proteins exhibit the most identities to

mammal selenium dependent GPX4. However, plant genes
carry a codon for a cysteine residue instead of the opal codon
UGA used for insertion of a selenocysteine in mammal
GPXs. The selenocysteine residue is important for the
catalytic activity of GPX as replacement of selenocysteine by
cysteine greatly reduces the activity of the enzyme [12].
According to Eshdat et al. [13], this would result in a plant
activity lower by three orders of magnitude when compared
to the homologous animal GPX. However, replacement of
the cysteine by a selenocysteine residue in the citrus GPX
was not followed by a gain in activity comparable to that
observed with selenium-dependent animal GPX [14]. Thus,
the physiological role of plant GPXs is not yet clear.
Furthermore, emerging reports on different living organisms
display opposite results about the enzymatic functions of
these GPX-like proteins [15–18]. These data prompted us to
explore the enzymatic functions of these proteins in higher
plants. In the present study, we characterized the expression
in E. coli of two plant GPXs, GPXle1 and GPXha2, from
Lycopersicon esculentum and Helianthus annuus, respect-
ively. The purified recombinant proteins were obtained and
used in enzymatic assays with various substrates in order to
investigate their putative function.
Correspondence to P. Roeckel-Drevet, UMR 547-PIAF INRA/
Universite
´
Blaise Pascal, 24 avenue des Landais, 63177 Aubie
`
re,
France.

Fax:+33473407916,Tel.:+33473407912,
E-mail:
Abbreviations: ROS, reactive oxygen species; GPX, glutathione
peroxidase; GSH, glutathione; PHGPX, phospholipid hydroperoxide
glutathione peroxidase; TPX, thioredoxin peroxidase.
Enzymes: catalase (EC 1.11.1.6); glutathione peroxidase (EC 1.11.1.9);
glutathione reductase (EC 1.6.4.2); glutathione-S-transferase
(EC 2.5.1.18);
L
-ascorbate peroxidase (EC 1.11.1.11); thioredoxin
reductase (EC 1.6.4.5); superoxide dismutase (EC 1.15.1.1).
(Received 2 January 2002, revised 19 March 2002,
accepted 26 March 2002)
Eur. J. Biochem. 269, 2414–2420 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02905.x
MATERIALS AND METHODS
Plant materials and chemicals
Tomato (Lycopersicon esculentum Mill. cv. VFN8) and
sunflower plants (Helianthus annuus Hybrid EL64, kindly
provided by F. Vear, INRA, Clermont-Ferrand, France)
were raised from seeds in moist vermiculite in a controlled
environment room: 16 h daylight at 60 lmolÆm
)2
Æs
)1
,
photosynthetically active radiation provided by 40-W white
daylight tubes (Mazda LDL, TF 40), 23 ± 1 °C(day)and
19 ± 1 °C (night), 60 ± 10% relative humidity. At the
cotyledon stage, tomato plants were transferred to a mineral
solution [19], while sunflower plants were grown in pots.

Glutathione, Saccharomyces cerevisiae glutathione reduc-
tase, b-NADPH, E. Coli thioredoxin, E. Coli thioredoxin
reductase, Triton X-100 (peroxide free), t-butyl hydroper-
oxide, cumene hydroperoxide, hydrogen peroxide, linoleic
acid,
L
-a-phosphatidylcholine dilinoleoyl and soybean
lipoxidase (type IV) were purchased from Sigma (Saint
Quentin Fallavier, France). Linoleic acid and
L
-a-phos-
phatidylcholine dilinoleoyl hydroperoxides were prepared
using soybean lipoxidase as described previously [20].
Hydroperoxides formation was monitored by following
the change in absorbance at 234 nm and their concentration
calculated using an e-value of 25 000
M
)1
Æcm
)1
.The
hydroperoxides were stored in ethanol at )20 °C.
Heterologous expression and purification
of recombinant GPXle1 and GPXha2
Total RNA was extracted from Lycopersicon esculentum
internodes and Helianthus annuus leaves according to the
method of Hall [21]. Full-length cDNAs encoding GPXle1
(GenBank accession number y14762) and GPXha2 (Gen-
Bank accession number y14707) were amplified by reverse-
transcription and PCR amplification as described by Drevet

et al. [22] using total RNA as template. During amplifica-
tion, the cDNAs were tagged with NdeI sites using
appropriate primers. The sequence of the primers used in
this study were 5¢-GAATTCGACATATGGCTACGC-3¢/
5¢-GCTCTCCCATATGGTCG-3¢ and 5¢-CGATAAGCA
TATGGCTACGC-3¢/5¢-GAATACTCAACATATGCAT
CC-3¢ for each set of forward/reverse gpxle1 and gpxha2
primers, respectively. Amplified products were subsequently
cloned into the NdeI linearized pET15b vector (Novagen,
Fontenay-sous-bois, France) at the NdeI site to give
in-frame fusion with a His
6
tag, and transformed in E. coli
BL21 (DE3) pLysS (Promega, Charbonnieres, France). For
both clones, sequence fidelity and proper insertion were
checked out by automated dye terminator sequence analysis
using the CEQ 2000 sequencer (Beckman-Coulter, Roissy
Charles De Gaulle, France). Clones were grown in ampi-
cillin (100 mgÆL
)1
)-supplemented Luria–Bertani media at
37 °CuptoD
600
¼ 0.6 and induced with 0.5 m
M
isopro-
pyl thio-b-
D
-galactoside. Four hours after induction, cells
were harvested by centrifugation (5000 g,10 min,4 °C) and

resuspended in 0.05
M
sodium phosphate, 0.3
M
NaCl,
0.02
M
imidazole at pH 7.5. The cells were then disrupted
by sonication at 10 kHz for a total of 60 s with five intervals
of 20 s each, and cell debris were sedimented by centrifu-
gation (10 000 g,30min,4°C). The presence of the
expected soluble recombinant protein was ascertained by
SDS/PAGE. The His-tagged protein products of GPXle1
and GPXha2 were affinity purified from cell extracts on
Ni
2+
-nitrilotriacetic acid matrix column according to the
manufacturer’s instructions (Qiagen, Courtaboeuf, France).
Protein concentrations in the eluted fractions were deter-
mined using the Bradford assay [23] and fractions contain-
ing the protein peaks were assayed immediately for
enzymatic activity. As a control, cultures of E. coli BL21
(DE3) pLysS transformed with pET15b vector alone were
treated as indicated above in parallel experiments.
Enzymatic assays
Glutathione-dependent peroxidase activity was measured
by monitoring NADPH oxidation with spectrophotometry
at 340 nm [24]. A standard reaction mixture (1 mL),
containing 100 m
M

Tris/HCl, pH 7.5, 5 m
M
EDTA,
0.2 m
M
b-NADPH, 3 m
M
GSH, 0.1% (v/v) triton X-100,
1.4 U of glutathione reductase and 50–100 lg of recom-
binant protein, was incubated at 30 °Cfor5min.After
3 min of equilibration, the reaction was initiated by the
addition of the peroxide substrate. The nonenzymatic
activity due to auto-oxidation of GSH as well as the activity
of any potentially co-purified E. coli proteins were also
examined. Corrections were made to estimate the activity of
recombinant proteins per se. Enzyme activities were calcu-
lated using an e-value of 6220
M
)1
Æcm
)1
. For measurement
of thioredoxin-dependent peroxidase activity, GSH and
glutathione reductase in the above-mentioned mixture were
replaced with E. coli thioredoxin (4 l
M
)andE. coli thiore-
doxin reductase (0.3 UÆmL
)1
), respectively. NADPH-

dependent peroxidase activity was assayed in a similar
fashion to glutathione-dependent peroxidase activity, except
that GSH and glutathione reductase were not added to the
reaction mixture.
RESULTS
Heterologous expression of GPXle1 and GPXha2
E. coli BL21 (DE3) pLys cells transformed with the
pET15b-derived expression plasmid efficiently produced
GPXle1 or GPXha2, as indicated by the presence of a
prominent band slightly greater than 20 kDa using SDS/
PAGE [Fig. 1]. This apparent molecular mass was in
agreement with the expected molecular mass (2181 Da from
the His
6
tag plus 18 847 Da from GPXle1 or 19 175 Da
from GPXha2). The purification scheme using Ni-nitrilo-
triacetic acid affinity matrix yielded a product of apparent
electrophoretic homogeneity (Fig. 1). No product was
purified from extracts from E. coli transformed with
pET15b vector alone (data not shown).
Enzymatic properties of GPXle1 and GPXha2
The glutathione peroxidase activities of GPXle1 and
GPXha2 towards several physiological and nonphysiolog-
ical hydroperoxides were monitored in the presence of
glutathione and glutathione reductase. Assays were carried
out using purified recombinant proteins or, as negative
controls, using either extracts from E. coli transformed with
the pET15b vector alone that had been affinity purified in
parallel or elution buffer alone. Such controls accounted for
Ó FEBS 2002 Dual activities for plant antioxidant enzymes (Eur. J. Biochem. 269) 2415

any nonenzymatic background due to auto-oxidation of
GSHandalsoanyE. coli peroxidase activity that might
have co-purified with the recombinant proteins. We found
no difference between NADPH oxidation in the presence of
affinity purified extracts from the E. coli control or in the
presence of the elution buffer alone. These data suggested
that no E. coli peroxidase activity copurified with our
recombinant proteins. Under these conditions, the apparent
K
m
and V
max
values for a variety of substrates were
calculated for GPXle1 and GPXha2 (Table 1). Both
proteins exhibited a higher affinity towards phospholipid
hydroperoxides and a weaker affinity towards t-butyl
hydroperoxide, as indicated by their respective apparent
K
m
values. There was no detectable activity with hydrogen
peroxide.
Considering the replacement of the selenocysteine, one of
the catalytic residues known to be critical for animal GPX
activity, and considering the sequence identities with
PHGPX (GPX4) that was reported to have no specificity
towards GSH [25], we have investigated the electron donor
requirements of GPXle1 and GPXha2. Three alternative
physiological reducing substrates, GSH, thioredoxin and
NADPH, were tested. Peroxidase activities were assayed
with a fixed t-butyl hydroperoxide concentration (100 l

M
)
using four to five different reducing substrate concentra-
tions. As carried out for GPX activity, control assays
accounted for any nonenzymatic NADPH oxidation and
also for any co-purified E. coli peroxidase activity. A
thioredoxin-dependent peroxidase activity was found for
both recombinant proteins in addition to the GPX activity.
Double reciprocal plots of 1/activity against 1/[GSH]
(Fig. 2A) or 1/[thioredoxin] (Fig. 2B) were linear and
reproducible in each case. Under these conditions, apparent
K
m
and V
max
values were calculated (Table 2). Neither
GPXle1 nor GPXha2 were able to reduce t-butyl hydro-
peroxide (Table 2) or others peroxides (data not shown)
using NADPH as reducing substrate. Both plant enzymes
showed higher affinity by three orders of magnitude
towards E. coli thioredoxin than to GSH, as indicated
by apparent K
m
values. Moreover, in reducing t-butyl
hydroperoxide, apparent V
max
values revealed a thioredoxin-
dependent peroxidase activity fivefold higher than glutathi-
one-dependent peroxidase activity. For both proteins, the
catalytic efficiencies (V

max
/K
m
) in the presence of thiore-
doxin are a lot higher than in the presence of glutathione
[Table 2]. Thus, recombinant GPXle1 and GPXha2 pre-
sented a TPX activity, albeit a slight GPX activity. Substrate
specificities of the TPX activity was further investigated
using a fixed concentration of E. coli thioredoxin (4 l
M
)
and four to five different substrate concentrations (Table 3).
In agreement with the above data, whichever the tested
substrate, TPX activity was found to be greater than the
GPX activity in terms of efficiency and substrate affinity
(Tables 1 and 3). Furthermore, both enzymes were able to
reduce hydrogen peroxide, as well as linoleic acid, phos-
phatidylcholine dilinoleoyl and t-butyl hydroperoxides,
using thioredoxin as reducing substrate whereas such an
activity was not detected in the presence of GSH.
DISCUSSION
An increasing number of proteins having at least two
functions has been reported [26]. Among the GPX family,
Fig. 1. Analysis by SDS/PAGE of the recombinant GPXle1 and
GPXha2 proteins expressed in E. c oli cells and purified by Ni-nitrilo-
triacetic acid affinity. Each crude extract (10 lgofprotein)andpurified
recombinant enzyme (1 lgofprotein)wereanalyzedby15%SDS/
PAGE.Lane1,pET/GPXle1-transformedE. coli;lane2,purified
recombinant GPXle1; lane 3, pET/GPXha2-transformed E. coli;lane
4, purified recombinant GPXha2. Proteins were stained with

Coomassie brilliant blue. Positions and sizes of molecular mass protein
markers are shown on the left side of the panel.
Table 1. Glutathione peroxidase activities of GPXle1 and GPXha2 towards different substrates. Glutathione peroxidase assays were performed as
described in Experimental procedures with a fixed concentration of GSH (3 m
M
) using four or five different concentrations of peroxide. The data
were analyzed by a Linewaever–Burk representation. Apparent maximum velocities (App. V
max
), apparent maximum Michaelis constant (App.
K
m
) values (± SEM) and V
max
/K
m
ratios are shown as the average of three independent experiments. The Cit-sap protein values were taken from
reference [32]. LA-OOH, linoleic acid hydroperoxide; PCdili-OOH, phosphatidylcholine dilinoleoyl hydroperoxide; t-butyl-OOH, ter-butyl
hydroperoxide.
Substrate
GPXle1 GPXha2 Cit-Sap (citrus)
App. V
max
(nmolÆmin
)1
Æmg
)1
)
App. K
m
(l

M
) V
max
/K
m
App. V
max
(nmolÆmin
)1
Æmg
)1
)
App. K
m
(l
M
) V
max
/K
m
App. V
max
(nmolÆmin
)1
Æmg
)1
)
H
2
O

2
0––0 ––0
t-Butyl-OOH 37.7 ± 2.71 128 0.294 27.1 ± 1.45 95.3 ± 2.56 0.284 24
Cumene-OOH 57.5 ± 1.27 119.0 ± 1.39 3.03 38.9 ± 0.61 60.8 ± 1.37 0.640 50
LA-OOH 27.7 ± 0.04 39.3 ± 0.03 0.705 42.4 ± 0.11 82.7 ± 3.47 0.516 44
PCdili-OOH 19.0 ± 0.44 24.9 ± 0.82 0.763 15.8 ± 0.44 12.1 ± 0.55 1.31 40
2416 S. Herbette et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the animal GPX4 (PHGPX) has been reported to be both a
structural protein and an active enzyme in sperm cells [27].
In addition, the animal selenium-independent and epididy-
mis-restricted GPX (GPX5) was also recently suspected to
bear dual-function [8,28].
This report shows that the two previously reported plant
GPX-like proteins [10,11] display a thioredoxin-dependent
peroxidase activity as well as a glutathione peroxidase
activity. Based on identities in their primary sequences with
animal GPXs, they were found to be more related to GPX4,
the phospholipid hydroperoxide glutathione peroxidase
[10,11]. This is also the case for other characterized plant
GPXs [13]. The mammalian GPX4 differs from the other
animal GPXs in that the protein is monomeric due to
deletions in regions thought to mediate tetramerization [25].
The small size and hydrophobic surface of these proteins
can explain that PHGPXs (GPX4) are unique in their
activity towards hydroperoxides integrated in membranes
[29], suggesting that they may play a significant role in
protecting membranes from oxidative damage. The
sequence similarities led us to suggest that GPX4-like plant
GPXs could be involved in membrane protection. Indeed, in
our experiments, GPXle1 and GPXha2 were found to

display glutathione-dependent peroxidase activity towards
organic peroxides such as phospholipid hydroperoxides, but
not towards hydrogen peroxide, thus behaving as expected
for a GPX4-like GPX. However, these in vitro activities
remain low. This can be explained by the lack of the rare
selenocysteine residue replaced by a cysteine in the catalytic
site of plant GPXs [13]. To date, low activities [15,17,18], or
no activity [16,30], were recorded for all seleno-independent
GPXs that have been investigated. In addition, GPXle1 and
GPXha2 exhibit a low affinity towards GSH and present
apparent maximum velocities with glutathione concentra-
tions which are far above evaluated physiological values
estimatedtorangefrom1to4.5m
M
in the chloroplast [31].
Heterologous expressions of GPXle1 and GPXha2 in
E. coli do not seem to affect their activity, because in vitro
values were found to be similar to those obtained from a
plant purified citrus GPX [32]. The low PHGPX activity of
GPXle1 and GPXha2 recorded in vitro does not necessarily
reflect the in vivo situation and does not rule out the
possibility that these proteins are indeed involved in
phospholipid hydroperoxides detoxification in the cell. In
yeast, it has been reported that PHGPX deletion mutants
were sensitive to induced lipid peroxidation, suggesting that
this seleno-independent protein protects membranes from
oxidative stress [17].
Our in vitro analysis of GPXle1 and GPXha2 enzymatic
functions strongly suggests that these two GPXs can also
function as thioredoxin peroxidases (TPX). Such a finding

was recently reported for a previously characterized GPX
from Plasmodium falciparum, which as a consequence has
been reclassified as a TPX [18]. In addition, it has been very
recently shown that a protein from chinese cabbage, which
is highly homologous to PHGPX, functions also as a TPX
[33]. Dual function for an antioxidant enzyme has also been
recently reported for a human 1-cys peroxiredoxin, which
exhibits glutathione peroxidase activity [34], and a bovine
eye protein showing homologies to TPXs but acting as a
seleno-independent GPX [35]. Our TPX assays rely on the
use of exogenous thioredoxin and thioredoxin reductase
from E. coli, instead of Lycopersicon esculentum and
Helianthus annuus endogenous ones. This bacterial thiore-
doxin system has successfully been used with the plasmo-
dium TPX protein [18]. As it was the case with the TPX
from Plasmodium falciparum, one could expect that GPXle1
and GPXha2 react faster with endogenous thioredoxins
from their respective plant species. However, thioredoxin
systems are probably not markedly different among living
organisms as proved by the fact that an E. coli thioredoxin
has been shown to enhance recovery of human cells after
Fig. 2. Analysis of GPXle1- and GPXha2-catalyzed reduction of t-butyl
hydroperoxide (100 l
M
) with different concentrations of GSH (A) and
thioredoxin (B). The reciprocal apparent maximum velocities of
GPXle1 (d)andGPXha2(s) are plotted against the reciprocal GSH
concentrations (1–10 m
M
)orE. coli thioredoxin concentrations

(1–6 l
M
) as a Linewaever–Burk representation. Each value (± SEM)
is representative of three experiments. GPX and TPX activities are
expressed as nmol of NADPH oxidized per min per mg of protein, and
GSH concentrations are expressed in m
M
whereas thioredoxin con-
centrations are expressed in l
M
.
Ó FEBS 2002 Dual activities for plant antioxidant enzymes (Eur. J. Biochem. 269) 2417
oxidative stress [36]. Thus, it is likely that the TPX activities
recorded in the present study reflect the activity in plants as
well. Supporting further this dual GPX/TPX function,
sequence alignments have shown that amino-acid residues
necessary for GSH specificity are not conserved in plasma
GPX (GPX3) and PHGPX (GPX4) groups (which include
GPXle1 and GPXha2), suggesting that GSH is unlikely to
be the sole physiological electron donor under all circum-
stances. For example, GPX3 can use thioredoxin as a
reducing substrate [37], and GPX4 exhibits an alternate
enzymatic thiol oxidase activity towards thiols contained in
various proteins [38]. Nevertheless, GPXle1 and GPXha2
do not accept all reducing substrates, as indicated by the
lack of activity when NADPH (Table 2) or NADH (data
not shown) were used. This implies that GSH and
thioredoxin affinities are somehow specific. Altogether,
these data on plant and animal GPXs suggest a putative link
existing between the glutathione-based antioxidant system

and the thioredoxin-based one.
TPX activities monitored here can be considered physio-
logical. Indeed, apparent K
m
values for thioredoxin are of a
micromolar range, compatible with in vivo levels. An in vivo
competition between GSH and thioredoxin for the plant
GPXs cannot be ruled out, because of the uncertainties
about the ratio between GSH and thioredoxin concentra-
tions in many tissues and physiological circumstances. In
line with these considerations, we can assume that the
electron donor and therefore the enzymatic function of the
proteins would depend on this ratio. Although there is no
sequence homologies between our plant GPXs and classical
TPXs, some similarities can be found with the PHCC-TPx
from chinese cabbage [33]. In particular, there are several
Cys residues that can be found at roughly equivalent
positions in these proteins. Interestingly, Jung et al.[33]
have put forward a putative role played by Cys residues in
the dual GPX/TPX catalytic process (i.e. exchange of
disulfide bonds) in the chinese cabbage PHCC-TPx. In any
case, the efficient TPX activity of GPXle1 and GPXha2 do
not exclude a PHGPX function but rather points to other
unknown biological roles for plant PHGPXs. Another
interesting trait of our results is that both GPXle1 and
GPXha2 can reduce hydrogen peroxide in the presence of
thioredoxin but not in the presence of GSH. Such data are
in agreement with the literature, as classical TPXs [39] are
known to metabolize hydrogen peroxide while plant GPX-
like enzymes do not [29].

This report shows that in plants, GPXle1 and GPXha2
can behave in vitro both as a GPX or/and as a TPX,
provided that the proper substrate and electron donor are
available. Considering the various subcellular localizations
of plant PHGPX-like proteins [10,11,40,41], the variations
in the tissue and the subcellular concentrations of substrates
and reducing substrates, dual catalytic activities for a given
enzyme might constitute an economical way plant cells have
evolved in order to cope with various physiological stresses
or situations. Indeed, we have previously shown that both
biotic and abiotic stresses were able to increase GPXha2
expression at the mRNA level [11].
Further in vivo investigations such as mutant analysis or
modifications of expression in transgenic plants will be
necessary to clarify this dual physiological role of plant
PHGPXs.
Table 3. Thioredoxin-dependent peroxidase activities of GPXle1 and GPXha2 towards different substrates. Thioredoxin-dependent peroxidase assays
were performed as described in Experimental procedures with a fixed concentration of E. coli thioredoxin (4 l
M
) using four different peroxide
concentrations. The data were analyzed by a Linewaever–Burk representation. Apparent maximum velocities (App. V
max
), apparent maximum
Michaelis constant (App. K
m
) values (± SEM) and V
max
/K
m
ratios are shown as the average of three independent experiments. LA-OOH, linoleic

acid hydroperoxide; Pcdili-OOH, phosphatidylcholine dilinoleoyl hydroperoxide; t-butyl-OOH, tert-butyl hydroperoxide.
Substrate
GPXle1 GPXha2
App. V
max
(nmolÆmin
)1
Æmg
)1
)
App. K
m
(l
M
) V
max
/K
m
App. V
max
(nmolÆmin
)1
Æmg
)1
)
App. K
m
(l
M
) V

max
/K
m
H
2
O
2
153.8 ± 1.79 13.7 ± 0.02 11.2 147.1 ± 2.12 13.9 ± 0.20 10.6
t-Butyl-OOH 147.2 ± 1.34 16.6 ± 0.28 8.87 161.3 ± 0.96 14.1 ± 0.35 11.4
LA-OOH 147.1 ± 1.11 8.60 ± 0.50 17.1 169.5 ± 1.46 16.2 ± 0.35 10.5
PCdili-OOH 108.7 ± 0.56 14.4 ± 0.12 7.55 126.7 ± 0.05 9.44 ± 0.31 13.4
Table 2. Reducing substrate specificities of GPXle1 and GPXha2 in catalyzed reduction of t-butyl hydroperoxide (100 l
M
). Peroxidase assays were
performed as described in experimental procedures with a fixed concentration of t-butyl hydroperoxide (100 l
M
) using four or five different
reducing substrate concentrations. The reducing substrates tested are GSH (1–10 m
M
), NADPH (100–200 l
M
)andE. coli thioredoxin (1–6 l
M
).
The data were analyzed by a Linewaever–Burk representation as illustrated in Fig. 2. Apparent maximum velocities (App. V
max
), apparent
maximum Michaelis constant (App. K
m
)values(±SEM)andV

max
/K
m
ratios are shown as the average of three independent experiments.
Substrate
GPXle1 GPXha2
App. V
max
(nmolÆmin
)1
Æmg
)1
)
App. K
m
(l
M
) V
max
/K
m
App. V
max
(nmolÆmin
)1
Æmg
)1
)
App. K
m

(l
M
) V
max
/K
m
Glutathione 48.8 ± 4.56 9300 ± 209 5.24 · 10
)3
46.7 ± 3.89 4900 ± 120 9.53 · 10
)3
NADPH 0 – – 0 – –
Thioredoxin (E. coli) 263.2 ± 0.36 2.2 ± 0.30 119.6 243.9 ± 0.50 1.5 ± 0.06 162.6
2418 S. Herbette et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENTS
S. H. is a recipient of a french pre-doctoral fellowship (Ministe
`
re de la
Recherche et de l’Enseignment Supe
´
rieur). We thank G. Pe
´
riot for
technical assistance and Dr E. Mare
´
chal (Laboratoire de Physiologie
Cellulaire Ve
´
ge
´
tale, CEA, Grenoble, France) for the gift of the pET15b

vector.
REFERENCES
1. Alsher, R.G. & Hess, J.L. (1993) Antioxydants in Higher Plants.
CRC Press, Boca Raton, FL.
2. Foyer, C.H. & Mullineaux, P. (1994) Causes of Photooxidative
Stress and Amelioration of Defense Systems in Plants. CRC Press,
Boca Raton, FL.
3. Dangl, J.L., Dietrich, R.A. & Richberg, M.S. (1996) Death don’t
have no mercy: cell death programs in plant–microbe interactions.
Plant Cell 8, 1793–1807.
4. Hammond-Kosack, K.E. & Jones, J.D. (1996) Resistance gene-
dependent plant defence responses. Plant Cell 8, 1773–1791.
5. Lamb, C. & Dixon, R.A. (1997) The oxidative burst in plant
disease resistance. Annu.Rev.PlantMol.Biol.48, 89–108.
6. Levine, A., Tenhaken, R., Dixon, R. & Lamb, C. (1994) H
2
O
2
from the oxidative burst orchestrates the plant hypersensitive
disease resistance response. Cell 79, 583–593.
7. Chu, F.F. (1994) The human glutathione peroxidase genes GPX2,
GPX3 and GPX4 map to chromosome 14, 5 and 19, respectively.
Cytogenet. Cell. Genet. 66, 96–98.
8. Drevet, J.R. (2000) Glutathione peroxidases expression in
the mammalian epididymis and vas deferens. In Proceedings
of the 1st European Congress of Andrology (Francavilla, F.,
Francavilla, S. & Forti, G., eds), pp. 427–461. Andrology 2000,
Aquila, Italy.
9. Holland, D., Ben-Hayyim, G., Faltin, Z., Camoin, L., Strosberg,
A.D. & Eshdat, Y. (1993) Molecular characterization of salt-

stress-associated protein in citrus: protein and cDNA sequence
homology to mammalian glutathione peroxidases. Plant Mol.
Biol. 21, 923–927.
10. Depege, N., Drevet, J. & Boyer, N. (1998) Molecular cloning and
characterization of tomato cDNAs encoding glutathione perox-
idase-like proteins. Eur. J. Biochem. 253, 445–451.
11. Roeckel-Drevet, P., Gagne, G., Tourvieille de Labrouhe, D.,
Dufaure, J.P., Nicolas, P. & Drevet, J.R. (1998) Molecular char-
acterization, organ distribution and stress-mediated induction
of two glutathione peroxidase-encoding mRNAs in sunflower
(Helianthus annuus). Physiol. Plant 103, 385–394.
12. Maiorino, M., Aumann, K.D., Brigelius-Flohe, R., Doria, D., van
denHeuvel,J.,McCarthy,J.,Roveri,A.,Ursini,F.&Flohe,L.
(1995) Probing the presumed catalytic triad of selenium-contain-
ing peroxidases by mutational analysis of phospholipid hydro-
peroxide glutathione peroxidase (PHGPx). Biol. Chem. Hoppe
Seyler 376, 651–660.
13. Eshdat, Y., Holland, D., Faltin, Z. & Ben Hayyim, G. (1997) Plant
glutathione peroxidases. Physiol. Plant 100, 234–240.
14. Hazebrouck, S., Camoin, L., Faltin, Z., Strosberg, A.D. & Eshdat,
Y. (2000) Substituting selenocysteine for catalytic cysteine 41
enhances enzymatic activity of plant phospholipid hydroperoxide
glutathione peroxidase expressed in Escherichia coli. J. Biol. Chem.
275, 28715–28721.
15. Wilkinson, S.R., Meyer, D.J. & Kelly, J.M. (2000) Biochemical
characterization of a trypanosome enzyme with glutathione-
dependent peroxidase activity. Biochem. J. 352, 755–761.
16. Gaber, A., Tamoi, M., Takeda, T., Nakano, Y. & Shigeoka, S.
(2001) NADPH-dependent glutathione peroxidase-like proteins
(Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in

Synechocystis PCC 6803. FEBS Lett. 499, 32–36.
17. Avery, A.M. & Avery, S.V. (2001) Saccharomyces cerevisiae
expresses three phospholipid hydroperoxide glutathione perox-
idases. J. Biol. Chem. 276, 33730–33735.
18. Sztajer, H., Gamain, B., Aumann, K.D., Slomianny, C., Becker,
K., Brigelius-Flohe, R. & Flohe, L. (2001) The putative glu-
tathione peroxidase gene of Plasmodium falciparum codes for a
thioredoxin peroxidase. J. Biol. Chem. 276, 7397–7403.
19. Morizet, J. & Mingeau, M. (1976) Influence des facteurs du milieu
sur l’absorption hydrique. Etude effectue
´
e sur la tomate de
´
capite
´
e
en exsudation. Ann. Agron. (Paris) 27, 183–205.
20. Ursini, F., Maiorino, M. & Gregolin, C. (1985) The selenoenzyme
phospholipid hydroperoxide glutathione peroxidase. Biochim.
Biophys. Acta 839, 62–70.
21. Hall, T.C., Buchinder, M.Y., Pyres, J.W., Sun, S.M. & Bliss, F.A.
(1978) Messenger RNA for G1 protein of french bean seeds: cell
free translation and product characterization. Proc. Natl Acad.
Sci. USA 75, 3196–3200.
22. Drevet, J.R., Swevers, L. & Iatrou, K. (1995) Developmental
regulation of a silkworm gene encoding multiple GATA-type
transcription factors by alternative splicing. J. Mol. Biol. 246,
43–53.
23. Bradford, M.N. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding. Anal. Biochem. 72, 248–254.
24. Maiorino, M., Gregolin, C. & Ursini, F. (1990) Phospholipid
hydroperoxide glutathione peroxidase. Methods Enzymol. 186,
448–457.
25. Brigelius-Flohe, R., Aumann, K.D., Blocker, H., Gross, G., Kiess,
M., Kloppel, K.D., Maiorino, M., Roveri, A., Schuckelt, R.,
Usani, F. et al. (1994) Phospholipid-hydroperoxide glutathione
peroxidase. Genomic DNA, cDNA, and deduced amino acid
sequence. J. Biol. Chem. 269, 7342–7348.
26. Jeffery, C.J. (1999) Moonlighting proteins. Trends Biochem. Sci.
24, 8–11.
27. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A.,
Wissing, J. & Flohe, L. (1999) Dual function of the selenoprotein
PHGPX during sperm maturation. Science 285, 1393–1396.
28. Drevet, J.R. (2001) Regulation of gene expression in epididymis.
In Proceedings of the Vii
th
International Congress of Andrology
(Robaire, B., Chernes, H. & Morales, C., eds), pp. 199–213.
Medimond Medical Publications, Montre
´
al, Quebec, Canada.
29. Ursini, F. & Bindoli, A. (1987) The role of selenium peroxidases in
the protection against oxidative damage of membranes. Chem.
Phys. Lipids 44, 255–276.
30. Okamura, N., Iwaki, Y., Hiramoto, S., Tamba, M., Bannai, S.,
Sugita, Y., Synti, P., Dacheux, F. & Dacheux, J.L. (1997)
Molecular cloning and characterization of the epididymis-specific
glutathione peroxidase-like protein secreted in the porcine epidi-
dymal fluid. Biochim. Biophys. Acta 1336, 99–109.

31. Noctor, G. & Foyer, C.H. (1998) Ascorbate and glutathione:
keeping active oxygen under control. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 49, 249–279.
32. Beeor-Tzahar, T., Ben-Hayyim, G., Holland, D., Faltin, Z. &
Eshdat, Y. (1995) A stress-associated citrus protein is a distinct
plant phospholipid hydroperoxide glutathione peroxidase. FEBS
Lett. 366, 151–155.
33. Jung, B.G., Lee, K.O., Lee, S.S., Chi, Y.H., Jang, H.H., Kang,
S.S., Lee, K., Lim, D., Yoon, S.C., Yoon, D.J., Inoue, Y., Cho,
M.J. & Lee, S.Y. (2002) A Chinese cabbage cDNA with high
sequence identity to phospholipid hydroperoxide glutathione
peroxidases encodes a novel isoform of thioredoxin-dependent
peroxidase. J. Biol. Chem. 31, DOI: 10.1074/jbc.M110791200.
34. Chen, J.W., Dodia, C., Feinstein, S.I., Jain, M.K. & Fisher, A.B.
(2000) 1-Cys peroxiredoxin, a bifunctional enzyme with glu-
tathione peroxidase and phospholipase A2 activities. J. Biol.
Chem. 275, 28421–28427.
Ó FEBS 2002 Dual activities for plant antioxidant enzymes (Eur. J. Biochem. 269) 2419
35. Singh, A.K. & Shichi, H. (1998) A novel glutathione peroxidase in
bovine eye. Sequence analysis, mRNA level, and translation.
J. Biol. Chem. 273, 26171–26178.
36. Spector, A., Yan, G.Z., Huang, R.R., McDermott, M.J., Gasco-
yne, P.R. & Pigiet, V. (1988) The effect of H
2
O
2
upon thioredoxin-
enriched lens epithelial cells. J. Biol. Chem. 263, 4984–4990.
37. Bjo
¨

rnstedt, M., Xue, J., Huang, W., Akesson, B. & Holmgren, A.
(1994) The thioredoxin and glutaredoxin systems are efficient
electron donors to human plasma glutathione peroxidase. J. Biol.
Chem. 269, 29382–29384.
38. Godeas, C., Tramer, F., Micali, F., Soranzo, M., Sandri, G. &
Panfili, E. (1997) Distribution and possible novel role of phos-
pholipid hydroperoxide glutathione peroxidase in rat epididymal
spermatozoa. Biol. Reprod. 57, 1502–1508.
39. Chae, H.Z., Chung, S.J. & Rhee, S.G. (1994) Thioredoxin-
dependent peroxide reductase from yeast. J. Biol. Chem. 269,
27670–27678.
40. Churin, Y., Schilling, S. & Borner, T. (1999) A gene family
encoding glutathione peroxidase homologues in Hordeum vulgare
(barley). FEBS Lett. 459, 33–38.
41. Mullineaux, P.M., Karpinski, S., Jimenez, A., Cleary, S.P.,
Robinson, C. & Creissen, G.P. (1998) Identification of cDNAS
encoding plastid-targeted glutathione peroxidase. Plant J. 13,375–
379.
2420 S. Herbette et al. (Eur. J. Biochem. 269) Ó FEBS 2002