Glutathione peroxidase family – an evolutionary overview

Rogerio Margis1, Christophe Dunand2, Felipe K. Teixeira3,* and Marcia Margis-Pinheiro1,4

1 Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
2 Laboratory of Plant Physiology, University of Geneva, Switzerland
3 Departamento de Genética, Universidade Federal do Rio de Janeiro, Brazil
4 Departmento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Keywords
evolution; glutathione peroxidase;
peroxiredoxins; subcellular location;
thioredoxin
Correspondence
M. Margis-Pinheiro, Departamento de
Genética, Universidade Federal do Rio
Grande do Sul, Sala 207, Prédio 43312,
91501-970 Porto Alegre, Brazil
Fax: +55 51 3308 7311
Tel: +55 51 3308 9814
E-mail: marcia.margis@ufrgs.br
*Present address
Unité de Recherche en Génomique
Végétale, INRA-CNRS-UEVE, Evry Cedex,
France
(Received 4 March 2008, revised 20 May
2008, accepted 6 June 2008)
Glutathione peroxidases (EC 1.11.1.9 and EC 1.11.1.12) catalyze the reduc-
tion of H2O2 or organic hydroperoxides to water or corresponding alcohols
using reduced glutathione. Some glutathione peroxidase isozymes have a
selenium-dependent glutathione peroxidase activity and present a selenocy-
steine encoded by the opal TGA codon. In the present study, insights into
the evolution of the whole glutathione peroxidase gene family were
obtained after a comprehensive phylogenetic analysis using the improved
number of glutathione peroxidase sequences recorded in the PeroxiBase
database (http://peroxidase.isb-sib.ch/index.php). The identification of a
common ancestral origin for the diverse glutathione peroxidase clusters
was not possible. The complex relationships and evolutionary rates of this
gene family suggest that basal glutathione peroxidase classes, present in all
kingdoms, have originated from independent evolutionary events such as
gene duplication, gene losses, lateral gene transfer among invertebrates and
vertebrates or plants. In addition, the present study also emphasizes the
possibility of some members being submitted to strong selective forces that
probably dictated functional convergences of taxonomically distant groups.

doi:10.1111/j.1742-4658.2008.06542.x

Aerobic reactions lead to the accumulation of reactive
oxygen species, which can be toxic to the cells. Biotic
and abiotic stresses can trigger a dramatic increase in
the generation of reactive oxygen species such as super-
oxide radicals, hydroxyl radicals and hydrogen peroxide
in the intracellular environment. In this context, aerobic
organisms have developed several non-enzymatic and
enzymatic systems to neutralize these compounds. The
enzymatic systems include a set of gene products such as
superoxide dismutases, catalases, ascorbate peroxidases
and glutathione peroxidases (GPx) [1].
Glutathione peroxidase (EC 1.11.1.9 and EC
1.11.1.12) is the general name for a family of multiple
isozymes that catalyze the reduction of H2O2 or
organic hydroperoxides to water or corresponding
alcohols using reduced glutathione (GSH) as an elec-
tron donor (H2O2 + 2GSH fi GS-SG + 2H2O). In
Metazoa, some GPxs have a selenium-dependent gluta-
thione peroxidase activity, with selenocysteine being
encoded by an opal TGA codon [2,3]. In mammalian
tissues, there are four major selenium dependent GPx
isozymes: (a) classical GPx (GPx1), which is found in
red cells, liver, lung and kidney; (b) gastrointestinal
GPx (GPx2); (c) plasma GPx (GPx3), which is present
in different organs such as kidney, lung, epididymus,
vas deferens, placenta, seminal vesicle, heart and
muscle, and (d) phospholipid GPx (PHGPx4 or GPx4),
which is also broadly distributed in different tissues.
GPx1, 2 and 3 act as homotetramers, whereas GPx4 is
functional as a monomer [2]. GPxs also have distinct

Abbreviations
ER, endoplasmic reticulum; GPx, glutathione peroxidase; GSH, glutathione; PHGPx, phospholipid hydroperoxide glutathione peroxidase; Trx,
thioredoxin.

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Evolutionary overview of glutathione peroxidases
subcellular locations: GPx1 was identified in the cyto-
sol, nucleus and mitochondria; GPx2 accumulates in
the cytosol and nucleus; GPx3 is a secreted protein
also found in the cytosol, whereas GPx4 is present in
the nucleus, cytosol, mitochondria and bound to
membranes [3]. Two other isozymes, GPx5 and GPx6,
identified in mammals, are both closely related to
GPx3. However, GPx5 lacks the selenocysteine at the
active site and is secreted in epididymus [4]. GPx-6 was
identified in humans and pigs and is a selenium-depen-
dent GPx found in the olfactory epithelium [5].
More recently, a new phospholipid hydroperoxide
glutathione peroxidase (PHGPx) was described in
mammals, which incorporates cysteine instead of selen-
ocysteine in the conserved catalytic motif. This nonse-
lenocysteine PHGPx, also named NPGPx, corresponds
to the seventh group of GPx found in mammals. This
GPx has a molecular mass of approximately 22 kDa
with a low glutathione peroxidase activity in vitro. The
expression of NPGPx encoding gene has been con-
firmed in many tissues, including developing mammary
glands. NPGPx plays an essential role in breast cancer
cells in alleviating oxidative stress generated from poly-
unsaturated fatty acid metabolism [6].
Genes encoding PHGPx enzymes have been identi-
fied in many organisms [7–10]. PHGPx is unique in its
substrate specificity because it can interact with lipo-
philic substrates such as peroxidized phospholipids and
cholesterol, reducing them to hydroxide compounds
[11,12]. Animal PHGPxs, originally synthesized as a
precursor protein [13], have their N-terminal amino
acids processed and are subsequently targeted to mito-
chondrial and nuclear membranes.
Plant GPxs have been classified as the fifth group of
peroxiredoxins because they can reduce peroxide with
higher efficiency (sometimes exclusively) by the thiore-
doxin (Trx) system rather than by using GSH as a
reducing agent [14]. The same situation was verified
for the protozoa Plasmodium falciparum, where a GPx-
like protein was also specific for Trx [15]. The thiore-
doxin specificity of Drosophila melanogaster GPx was
also recently demonstrated [16]. At the molecular level,
plant GPx genes are closely related to animal PHGPx
and their corresponding proteins present three widely
conserved cysteine residues, which are assumed to be
essential for the enzymatic catalysis. In poplar, site-
directed mutagenesis indicates that only two Cys
(Cys107 and Cys155) among the three conserved resi-
dues, which form a disulfide bridge, participate in the
catalytic cycle and in Trx-mediated regeneration.
Moreover, the plant GPx gene family may comprise
up to six members that are distributed in different
subcellular compartments [17].
3960 FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS
R. Margis et al.
The extensive available data related to the structure
and function of animal, plant and fungal GPx repre-
sent an important achievement toward understanding
the function of this class of proteins. However, a phy-
logenetic study of the whole family of GPx proteins is
still lacking. To date, few studies have been published
describing independent phylogenetic studies of some
GPx classes [3,18]. The present study aimed to examine
the phylogenetic relationships among GPx of various
organisms and to contribute to the understanding of the
possible modes of evolution of this family. The results
obtained suggest that the evolutionary history of GPx
genes is highly complex and is the result of a number
of independent evolutionary events that might be in
association with processes of functional convergence.
Results
Phylogenetic relationships
Phylogenetic analyses were carried out with GPx
amino acid sequences from species of different phyla
representative of Viridiplantae, fungi and metazoan
kingdoms, protists and prokaryotes. The GPx gene
family has an uncertain origin and does not follow a
linear evolutionary history. It is apparent that different
and independent evolutionary pathways have operated
in their members, making it difficult to establish an
ancestral origin for many classes of GPx. However, it
is possible to visualize three main GPx clusters with
possible polyphyletic origins (Fig. 1). Group I corre-
sponds to a large cluster of all metazoan GPx, includ-
ing the seven classes originally described in mammals,
also extending to other groups of vertebrate (v1 to v7)
plus invertebrate GPxs sequences. In this group,
sequences from parasitic invertebrates (worms) clus-
tered with vertebrate GPxs from classes 1, 2, 3, 5 and
6, whereas arthropod sequences grouped closer to class
4 GPx. Group II encompasses GPx sequences from
fungi, bacteria, cyanobacteria and two algae sequences
(CreGPx03, SobGPx01), whereas group III comprises
all plant GPxs. The major part of algae GPxs clustered
as an independent and disperse group, more closely
related to the Kinetoplastida GPx sequences than to
the plant or bacterial sequences. Taken together, the
results suggest that all these proteins originated very
early in the evolutionary history of GPx genes. It is
possible that these proteins possess a common origin;
however, this is hard to determine because their
evolution is complex and ancient. One strategy for
obtaining a better understanding about GPx
evolutionary history consists of the analysis of each
main group separately.
R. Margis et al. Evolutionary overview of glutathione peroxidases

Fig. 1. Global phylogenetic tree of 204 GPx amino acid sequences present in the PeroxiBase database. The three main encircled clusters
correspond to vertebrates (v1 to v7) and invertebrates (group I), bacteria and fungi (group II) and plants (group III). The SPLITSTREE was built
after a CLUSTALX multi-alignment and a Neighboir-joining analysis using p-distance and complete deletion parameters with 2000 bootstraps to
assured reliability of all nodes (not indicated).

trees suggest that the sequence of Xenopus (XlGPx01)

Phylogenetic analysis of vertebrate GPxs
A closer view of the phylogenetic relationships
among vertebrate GPx genes reveals the presence of
four main structured groups supported by high val-
ues of bootstrap (Fig. 2). The same tree topology
was obtained for the main GPx groups by using
parsimony and maximum likelihood (Fig. 2A) or
Neighbour-joining (Fig. 2B). The first vertebrate
group (GPx1 ⁄ 2) is split into two other clusters, char-
acterized by the presence of the mammalian, bird
and fish genes encoding the intracellular proteins
GPx1 and GPx2. Parsimony and Neighbour-joining
is in the base of this group. However, this position
is ambiguous because maximum likelihood placed
XlGPx01 inside the GPx2 cluster (see supplementary
Fig. S1). The GPx1 cluster consists of 11 representa-
tives of mammalian GPx1 and the bird sequence
GgaGPx01, whereas the GPx2 contains eight mam-
malian GPx2, the bird GgaGPx02 and three
zebrafish sequences (DrGPx02a, DrGPx2b and
DrGPx02c). This suggests that the duplication of
GPx1 and GPx2 genes occurred before the diver-
gence of birds and mammals, but probably after the
emergence of amphibians and fishes.

FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS 3961
Evolutionary overview of glutathione peroxidases
A B
The second vertebrate group encompasses the mam-
mals GPx3, GPx5 and GPx6. In this group, fish
DrGPx03 and amphibian XlGPx03 sequences group
together but outside of the mammalian ⁄ bird cluster.
This position is well supported by a strong bootstrap,
suggesting that the two successive duplications leading
to the appearance of GPx3, GPx5 and GPx6 occurred
only in mammals (Figs 1 and 2). In birds, a single gene
encoding GPx3 was identified (GgaGPx03). Note that
parsimony and maximum likelihood revealed that
GPx5 and GPx6 members are much more related to
each other than to their GPx3 relatives (Fig. 2A).
The third and the fourth groups correspond to more
distant and strongly supported branches: the GPx4
3962 FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS
R. Margis et al.
Fig. 2. Phylogenetic tree reconstruction
from a CLUSTALX multi-alignment of 85 amino
acid sequences from animal GPxs. (A) Coa-
lescent tree from parsimony and maximum
likelihood analyses from 200 informative, 96
constant and 12 non-informative out of the
308 animal GPxs characters. Bootstrap val-
ues for parsimony (upper values) and maxi-
mum likelihood (lower values) are indicated
in branches sharing strict topology. (B) The
tree was built using Neighbour-joining, p-dis-
tance, pairwise deletion and 2000 boot-
straps to estimate interior branch
confidence probability. Branch values with
more than 70% confidence are indicated.
White, two gray and black circles indicate
the conservative clusters encompassing
GPx1 ⁄ 2, GPx3 ⁄ 5 ⁄ 6, GPx7 and GPx4,
respectively.
and the GPx7, respectively. Both contain close related
sequences from mammals, birds (GgaGPx04 ⁄ 07),
amphibians (XlGPx04 ⁄ 07) and fishes (DrGPx04 ⁄ 07).
Two sequences were also identified in mollusks (Mcal-
GPx04 and AcaGPx04) and one in Cnidaria
(HmetGPx). These sequences, together with arthropod
GPx sequences (Fig. 2A,B), are much more divergent
and appeared outside of the vertebrate cluster.
Phylogenetic analysis of fungi, bacteria and
algae GPxs
All available sequences from fungi and bacteria clus-
tered in the main GPx group II, apart from meta-
R. Margis et al. Evolutionary overview of glutathione peroxidases

zoan group I and plant group III (Fig. 1). The clustered in five main groups. The previously predicted
detailed phylogenetic relationships among bacteria, subcellular localizations of Arabidopsis, rice and poplar
fungi and algae GPx sequences, inferred from the GPxs [17] are in agreement with the clustering topol-
alignment of 95 amino acid sequences (Fig. 3), reveal ogy obtained for these sequences. The genes classified
that these GPxs split into three main clusters. The as cytosolic are more divergent and were placed out-
first one encompasses eukaryotic unicellular organ-
isms such as Lobosea (HveGPx01), Glaucocysto-
phycea (CyGPx01 and GnoGPx01 and 02) and
Apicomplexa (PfaGPx01 and TgGPx01). In addition,
some Diatoms (TpsGPx01 and 02) and green algae
(CreGPx02 and 04, GsuGPx01) clustered in this
group. The second group contains mostly bacteria
GPx, with representatives of Alphaproteobacteria,
Betaproteobacteria and Gammaproteobacteria form-
ing three clear distinct subgroups. An exception to
this rule is the cluster of Kinetoplastida. Blue–green
algae (cyanobacteria SYspGPx01) and various mem-
bers of Firmicutes have a scattered distribution
inside the proteobacteria group. The third group
clusters together all the sequences detected in fungi
(Pezizomycotina and Saccharomycotina from Asc-
omycota) with the exception of a sequence from
algae, which appears at the base of the cluster. The
presence of a sequence in Basidiomycota is marginal
and has been only detected in Ustilago maydis (Um-
GPx). The analysis clearly shows that members from
Ascomycota are distributed in three clusters: one
corresponding to the subphylum Pezizomycotina and
two others containing members from the subphylum
Saccharomycotina, probably as the result of a gene
duplication event.

Phylogenetic analysis of plant GPxs

A meticulous phylogenetic analysis was also carried
out including 166 GPx from plants (Fig. 4). The clado-
gram obtained by parsimony or maximum likelihood
methods exhibited similar topology to the Neighbour-
joining phylogenetic tree and revealed that plant GPxs

Fig. 3. Phylogenetic relationships among bacteria, fungi and algae

GPxs inferred from a CLUSTALX multi-alignment of 95 amino acid
sequences. Clusters encompassing groups of organisms sharing
common evolutionary origin are indicated by brackets: fungi from
subphylum Pezizomycotina and Saccharomycotina; members from
alpha-, beta- and gammaproteobacteria; and members from kine-
toplastida, apicomplexa and glaucocystophyces. Classes of organ-
isms with a single representative were also indicated. The tree was
built using the Neighbour-joining method. The tree is drawn in scale
proportional to the evolutionary distances calculated by the Poisson
correction method. Confidence probability of interior branch lengths
was estimated after 2000 replicates using the bootstrap test. All
positions containing gaps and missing data were eliminated, with
114 positions remaining in the final dataset.

FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS 3963
Evolutionary overview of glutathione peroxidases
side of the main group that encompasses four other
clusters (Fig. 4). Each cluster is represented by GPx
proteins localized or predicted to be localized at differ-
ent subcellular compartments: mitochondria, chloro-
plast, endoplasmic reticulum (ER) ⁄ cytosol or secreted.
Except for the group of secreted GPxs, at least one
representative of monocots and dicots is found within
each branch. The plant phylogenetic tree (Fig. 4)
clearly shows that mitochondrial GPxs from grasses
clustered in two separated groups. In the other
branches (ER ⁄ cytosolic, chloroplastic and cytosolic),
GPx from monocots clustered in single groups of
grasses, indicating that the evolution of these proteins
within these groups was independent (Fig. 4, circles).
The analysis of the position of GPx sequences from
gymnosperm and cryptogams in the plant phylogenetic
tree (Fig. 4) reveals that the paralogs from cytoplasm,
chloroplast and mitochondria were already present in
these organisms. The presence of gymnosperm and
cryptogam sequences in the base of these branches
indicates the existence of initial duplication at least
before the emergence of gymnosperm. In addition, the
absence of gymnosperm and cryptogam sequences in
ER ⁄ cytosolic and the secreted groups suggests that the
3964 FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS
R. Margis et al.
divergence leading to the emergence of these groups
occurred only in angiosperms (Fig. 4).
Detailed phylogenetic analysis of thiol peroxidases,
including peroxiredoxins and plant, fungi and animal
GPxs, revealed that plant GPx are more closely related
to human GPx4 than to fungal GPxs (Fig. 5). The
analysis showed that GPxs and peroxiredoxins form
two well supported clusters in spite of their great simi-
larity, in relation to their preferential substrate for
catalytic reaction.
Discussion
It has been postulated that, in mammals, the GPX gene
family would have evolved from a common gene ances-
tor by duplication events, followed by random mobili-
zation in the genome. In this scenario, the ancestor was
proposed to be represented by the GPx4 ⁄ PHGPx
sequence [3]. In addition, it is known that mammalian
PHGPx appears to be more closely related to GPxs from
various organisms than to all mammalian GPx types.
Hence, the PHGPx group must be considered as a phy-
logenetically ancient achievement of the GPx family. In
the present study, we carried out a phylogenetic analysis
Fig. 4. Phylogenetic tree inferred from a
CLUSTALX multi-alignment of 166 amino acid
sequences from plant GPxs using the
Neighbour-joining method. Five sequences
from green algae (Chlorophyta) were added
to the analyses and used as the out-group.
Branches corresponding to different subcel-
lular compartments are indicated according
to previous experimental data from the liter-
ature. Positions corresponding to four puta-
tive ancestral GPx duplication events are
indicated with numbers from 1 to 4. Circles
correspond to a cluster only containing
monocot representatives. Squares indicate
the basal position of members from Gymno-
sperms or cryptogams in the main
branches. The bootstrap consensus tree and
evolutionary distances were obtained using
Neighbour-joining, Poisson correction, pair-
wise deletion and 2000 bootstraps. A total
of 323 positions were present in the final
dataset.
R. Margis et al.
Fig. 5. Thiol peroxidases phylogenetic tree inferred from a CLUSTALX
multi-alignment. A total of 126 sequences were used, encompass-
ing animal (15), plant (22) and fungi (23) with their respective HGPx
orthologs, all members of human GPxs and representatives of per-
oxiredoxins. Animal, plant and fungus HGPx clusters were plotted
as compressed subtrees (triangles) to facilitate visualization. Tree
was built using Neighbour-joining, p-distance, pairwise deletion and
2000 bootstraps. Branch values with more than 70% confidence
are indicated.
involving more than 200 GPx sequences from very
diverse groups of organisms (Fig. 1). With this strategy,
we analyzed the evolutionary relationships existing
between different GPx encoding genes, especially aiming
to understand why GPx4 proteins are conserved from
fungi to mammals and to determine whether all GPx4s
have the same origin. Our analyses provide evidence for
recent evolutionary events that resulted in the molecular
radiation of this gene family but were unable to depict
more ancient ones. The analysis reveals that vertebrate
GPx1, 2, 3, 5 and 6 may have had a common phyloge-
netic origin, probably distinct from GPx4. Arthropod
GPxs formed a well supported distinct group, but are
closely related to the GPx4 cluster. The basal position of
these genes in the different trees makes it difficult to
identify the existence of a GPx4 precursor that was
already present before irradiation between vertebrates
and invertebrates (Fig. 2B) or their polyphyletic origin
(Fig. 2A). Another ancestral GPx should have lead to
the origin of the group encompassing plants (group III;
Fig. 1) that form an independent cluster. Fungal and
bacterial GPxs form a group distinct from the others
FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS
Evolutionary overview of glutathione peroxidases
(group II; Fig. 1). The clocklike model associated with
the maximum likelihood analysis was rejected at a sig-
nificance level of 5% irrespective of the outgroup used
to root the tree. The log-likelihood of the more complex
(no clock) tree was significantly increased, indicating
that GPx genes have evolved at different rates.
The vertebrate cluster of GPxs (Fig. 2) is in agreement
and corroborates previous phylogenetic studies [3].
However, previous phylogenetic evaluations did not
include GPx7 sequences, which represent another set of
vertebrate PHGPxs. Phylogenetic tree reconstruction
produced ambiguous results. Although parsimony and
maximum likelihood analyses grouped members from
GPx7 together with GPx1, 2, 3, 5 and 6 (Fig. 2A), the
Neighbour-joining tree revealed GPx7 proteins to be
more related to GPx4 (Fig. 2B). Our result suggests that
all vertebrate GPx gene families evolved by duplication
from a common ancestral gene probably related to the
PHGPx sequence because sequences related to this
group are also present in plants, arthropods and fungi.
After the first duplication event in vertebrates, this
ancestor diverged to form two groups: the first evolved
into GPx4 sequences, and the second group diverged
subsequently to produce the GPx7, GPx1 ⁄ GPx2 group
and the GPx3 ⁄ GPx5 ⁄ GPx6 cluster (Figs 1 and 2).
The clustering of GPx4 animal sequences together
with plant and fungi instead of other animal sequences
(Fig. 5) would suggest the existence of an evolutionary
convergence orchestred by functional pressures [19–23].
As previously demonstrated [3], our analysis confirmed
that mammalian PHGPx is more closely related to
GPx from various organisms than to all mammalian
GPx types (Figs 1 and 5). This result indicates that
PHGPx proteins have fundamental and conserved
functions in organism from different lineages, which
could strength the model of convergent evolution of
GPx genes. However, the conservation also suggests
that the precursor of this clad was already present in
the ancestral cells before the divergence between inver-
tebrates and vertebrates. Altogether, this global phylo-
genetic analysis could indicate that two different
ancestral GPx genes were present in the ancestral
organism. One sequence evolved only in the animal
lineage originated in the genes encoding GPx1 to
Gpx7. The second ancestral sequence originated in the
modern GPx4, currently present in different organisms
such as animals, plants and fungi. Alternatively, the
absence of GPx1, 2, 3, 5, 6 and 7 in plants and fungi
may have resulted from the loss of these genes during
evolution. In some nematodes and Ciona intestinalis,
the relationship with GPx4 could be explained by hori-
zontal gene transfer from vertebrates to these organ-
isms during parasitism or symbiosis [3,24].
3965
Evolutionary overview of glutathione peroxidases
Plant GPx and other thiol peroxidases
The global phylogenetic analysis showed that plant
GPxs form an independent cluster, suggesting that an
ancestral gene lead to the origin of all plant GPx
genes. The analysis of the phylogenetic relationships
between plants, yeasts and animals GPx4 (Fig. 5)
revealed that plant GPxs are more closely related to
animal GPx4s than to yeast GPxs. Recent studies
revealed that, similar to the yeast GPxs, the GPx fam-
ily of poplar can reduce peroxides by using Trx as a
reductant [17]. Hydroperoxide reduction by thioredoxin
was also demonstrated for Arabidopsis thaliana.
Recently, the crystal structure of reduced and oxidized
forms of poplar PtGPx05 was determined [25] and
revealed that this enzyme exhibits an overall structure
similar to the animal GPxs. The PtGPx05 possesses an
active site cleft architecture (consisting of Cys, Glu
and Trp) that is very similar to the mammal SeCys-
GPx structures, and distinguishes PtGPx05 from PRxs.
The authors suggest that PtGPx05 is a thioredoxin
peroxidase, being structurally related to glutathione
peroxidases but exhibiting catalytic and Trx-dependent
recycling mechanisms of peroxiredoxin. It is possible
that plant GPx could utilize a particular Trx or other
alternative reductant [18]. In Saccharomyces cerevisiae,
nonselenium-GPx reduces H2O2 using the transcription
factor YAP1 as reducing substrate [3,26]. Recently, it
was demonstrated that the Drosophila melanogaster
GPx exhibits a clear preference for Trx with a catalytic
mechanism involving the formation of internal disul-
fide that is pivotal to the interaction with Trx [16]. In
addition, molecular modeling and homology analysis
based on 450 GPx suggests that, in functional terms,
most of the nonmammalian NSGPxs are thioredoxin
peroxidases.
A reclassification of nonselenium-GPx away from
the GPx family has been proposed [3,16,27] based on
biochemical criteria because these enzymes use sub-
strates other than glutathione, especially thioredoxin.
The biochemical similarity among these enzymes was
not supported by our phylogenetic analyses because
these sequences clustered together with vertebrate
GPxs rather than with other thiol peroxidases. The
result demonstrated the complex evolutionary history
of GPx sequences, pointing to independent evolution-
ary pathways with the convergence of some lineages.
Sequence diversity and subcellular localization of
plant GPx
The phylogenetic tree of plant GPxs (Fig. 4) exhibits a
strong dichotomous structure clustering in separated
3966 FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS
R. Margis et al.
branch isoforms located at different subcellular local-
izations. Thus, isoforms belonging to the same subcel-
lular compartment are more related to each other than
to isoforms from the same organism but with a differ-
ent localization, demonstrating that proteins located at
the same subcellular compartment have a common
origin. Our results indicate that all plant GPx genes
were generated by four major duplication events from
a single ancestral gene and that these original duplica-
tions have occurred before the divergence of monocots
and dicots. The presence of paralogs in some branches
suggests that more recent duplications have occurred
independently in monocots and dicots (Fig. 4). Our
results also demonstrate that none of the monocot-
specific GPx clustered with any group of dicots,
suggesting that, after the radiation of monocots, GPx
evolved independently in both monocots and dicots.
Concerning the subcellular localization of plant
GPxs, it is likely that specific signatures for each sub-
group target them to different subcellular compart-
ments. In spite of this very well defined clustering of
plant GPx proteins, according to their predicted
location, only a few proteins were experimentally
demonstrated to be targeted to a specific subcellular
localization. A pea (Pisum sativum) GPx homologue to
the PtGPx01 was demonstrated to be imported to the
chloroplast [27]; tomato GPxle-1 and PtGPx05
(PtrGPx3.1) were found in the cytosol [3,17].
PtGPx06-1 (or PtrGPx3.2) is closely related to the
PtGPx06-2 (Fig. 4) clustering with other mitochondrial
proteins; however, this protein was experimentally
demonstrated to be targeted to both mitochondria
and chloroplasts [28]. It remains unknown whether
the GPx proteins, clustering in a same subcellular
compartment group, are effectively targeted to the
predicted location.
Comparative GPx gene organization
To investigate the relationships among the distinct
plant GPx and animal isoforms, the structural orga-
nization of GPx genes was performed by comparing
gene and cDNA sequences encoding the same iso-
form. All human, arabidopsis and rice GPx genes
were included in the analysis (Fig. 6). A detailed
comparison of GPx genes across plant species
revealed a high degree of conservation in gene struc-
ture. This suggests that the different GPx genes, cor-
responding to a protein with a distinct subcellular
location, were generated by duplication events from
a single nuclear ancestral gene. The organelle located
GPx may have originated later due to the gain of a
specific signature that targets these proteins to differ-
R. Margis et al.
ent locations. This contrasts with the results
observed for ascorbate peroxidase genes, which pres-
ent a high degree of conservation in gene structure
only for APx isoforms localized in the same subcel-
lular compartment. Moreover, the structure of
the APx genes encoding cytosolic and peroxisome
membrane-bound isoforms is very similar, differing
markedly from that of the chloroplastic and mito-
chondrial APx genes, and suggesting that an initial
duplication event generated the ancestral genes
encoding the chloroplastic and the nonchloroplastic
isoforms [29,30].
Human GPx genes revealed a high degree of conser-
vation in gene structure but only for GPxs from the
same cluster (Fig. 2). HsGPx01 and 02 present two
exons separated by a long intron, whereas HsGPx03, 05
and 06 contain five exons of almost the same size.
HsGPx05 can lead to the origin of two distinct tran-
scripts resulting from alternative splicing of the third
exon (Fig. 6). HsGPx04 and HsGPx07 present very dif-
ferent gene organizations, with seven and three exons,
respectively. Comparisons of the structural organiza-
tion of mammalian GPx genes further corroborate the
phylogenetic tree topology and reinforce the concept of
a common origin, with each isoform arising from a
common ancestor for mammalian GPx genes.
Plant GPx and human GPx4 gene organization is
very divergent, reinforcing the hypothesis of an
independent evolutionary history with functional con-
vergence. The analysis carried out in the present study
is important for understanding GPx evolution and
directing further functional studies. The mechanism of
GPx duplication is not clearly known. Their conserved
protein sequences, and known function, point to sub-
functionalization. Subfunctionalization is believed to
occur more frequently than neofunctionalization
because it exploits common degenerative mutations
rather than relying on rare beneficial mutations. How-
ever, neofunctionalization corresponding to the gain of
new functions cannot be discarded because the depen-
dence on thioredoxin as an electron donor instead of
glutathione in some GPx family members indicates
specialization towards a new function, as recently
demonstrated for PtGPx05 acting as a heavy metal
sink [25].
Experimental procedures
Searching for GPx sequences
Multiple database searches were performed to identify all
members of the GPx family in different organisms. The
majority of the protein sequences were obtained from the
FEBS Journal 275 (2008) 3959–3970 ª 2008 The Authors Journal compilation ª 2008 FEBS
Evolutionary overview of glutathione peroxidases
UniProt database (http://www.expasy.uniprot.org/). To
identify GPx sequences in a large variety of organisms,
GPx sequences from Arabidopsis were used as query
sequences in tBLASTn searches within different databases,
such as the NCBI website (http://www.ncbi.nih.gov/
BLAST) and TIGR (http://rice.plantbiology.msu.edu), and
in more specialized databases such as the Plant GDB
database (http://www.plantgdb.org/) and the DOE Joint
Genome Institute (JGI) website (http://www.jgi.doe.gov/).
The non-annotated sequences were analyzed for the
presence of a putative gene with two programs: fgenesh
(http://www.softberry.com/berry.phtml) and genscan
(http://genes.mit.edu/GENSCAN.html). The corresponding
coding sequences were translated with the ‘translate’ tool
available on the ExPASy proteomics server (http://us.
expasy.org/tools/dna.html) and further confirmed for the
existence of GPx motifs. All protein sequences described
and analyzed in the present study were manually annotated,
formatted and can be found in PeroxiBase, a public data-
base available at: http://peroxidase.isb-sib.ch [31,32]. The
numbering of new sequences was based on the existing
numbering of humans for Metazoan GPx, and rice and
Arabidopsis for monocot and dicot GPx, respectively.
Sequence and phylogenetic analyses
Among the available GPx sequences found in the different
databases, only complete protein sequences (or those cover-
ing more than 70% of their closer orthologs) representative
of each phylum were used to build a global phylogenetic
tree. Introduction of incomplete sequences could increase
the number of taxa represented in the analyses but would
also strongly deteriorate the phylogenetic analyses.
Sequence multiple-alignments were carried out using
clustalx (version 1.83) [33], with further visual inspection,
adjustment and realignments with clustalx.
Phylogenetic analyses using the Neighbour-joining
distance based method were performed with splitstree,
version 4.8 [34] or mega, version 4 [35], whereas charac-
ter-based methods were made with paup*, version 4.0
[36] and tree-puzzle, version 5.2 [37]. Both parsimony
and maximum likelihood analyses were used for tree
reconstruction of animal GPx amino acid sequences.
Maximum parsimony trees using paup* were obtained by
1000 random addition heuristic search replicates and with
the tree bisection–reconnection branch-swapping option.
Maximum likelihood tree reconstruction was made with
tree-puzzle using quartet sampling. Quartet puzzling
was used to choose the possible tree topologies and to
simultaneously infer support values for internal branches.
A total of 50 000 puzzling steps were run and 93% of the
2 024 785 analyzed quartets were fully resolved. Quartet
trees were based on approximate maximum likelihood val-
ues using the WAG model of amino acid substitution,
selected as the best lnL model out of five other models
3967
Evolutionary overview of glutathione peroxidases R. Margis et al.

Fig. 6. Organization of human, arabidopsis and rice GPx genes. Chromosomal localization of human (Hs), arabidopsis (At) and rice (Os) GPxs
are also indicated. The position and size of the intron ⁄ exons was predicted using FGENESH and GENSCAN. White boxes and lines correspond to
exons and introns, respectively. Numbers above each box ⁄ line indicate the intron ⁄ exon size. An asterisk indicates an exon absent in alterna-
tive splicing.

(Dayhoff, JTT, mtREV24, BLOSUM62, VT) and the
gamma distribution rate heterogeneity with an alpha equal
to 1.02, estimated from data set. A molecular clock
maximum likelihood ratio test, present in tree-puzzle, was
used to test whether GPx genes evolved according to the
molecular clock model. Phylogenetic trees and cladograms
were edited using treeview [38].
Acknowledgements
We thank Patricia Lariguet for her critical reading of
the article. M. M.-P. and R. M. were supported by
grants from Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico, CNPq, Brasil (308708 ⁄ 2006-7
and 302684 ⁄ 2005-0). The authors also thank the ano-
nymous referees for their critical comments that con-
tributed toward improving this manuscript.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Maximum likelihood phylogenetic tree of ani-
mal GPxs.
This material is available as part of the online article
from http://www.blackwell-synergy.com
Please note: Blackwell Publishing is not responsible
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