Báo cáo khoa học: Plasmoredoxin, a novel redox-active protein unique for malarial parasites doc

PRIORITY PAPER
Plasmoredoxin, a novel redox-active protein unique
for malarial parasites
Katja Becker
1
, Stefan M. Kanzok
2
, Rimma Iozef
1
, Marina Fischer
1
, R. Heiner Schirmer
2
and Stefan Rahlfs
1
1
Interdisciplinary Research Center, Justus-Liebig-University, D-35392 Gießen, Germany;
2
Biochemistry Center,
Ruprecht–Karls-University, D-69120 Heidelberg, Germany
Thioredoxins are a group of small redox-active proteins
involved in cellular redox regulatory processes as well as
antioxidant defense. Thioredoxin, glutaredoxin, and try-
paredoxin are members of the thioredoxin superfamily and
share structural and functional characteristics. In the mal-
arial parasite, Plasmodium falciparum, a functional thio-
redoxin and glutathione system have been demonstrated
and are considered to be attractive targets for antimalarial
drug development.
Here we describe the identification and characterization of
a novel 22 kDa redox-active protein in P. falciparum.As

demonstrated by in silico sequence analyses, the protein,
named plasmoredoxin (Plrx), is highly conserved but found
exclusively in malarial parasites. It is a member of the thio-
redoxin superfamily but clusters separately from other
members in a phylogenetic tree. We amplified the gene from
a gametocyte cDNA library and overexpressed it in E. coli.
The purified gene product can be reduced by glutathione but
much faster by dithiols like thioredoxin, glutaredoxin, try-
panothione and tryparedoxin. Reduced Plrx is active in an
insulin-reduction assay and reduces glutathione disulfide
with a rate constant of 640
M
)1
Æs
)1
at pH 6.9 and 25 °C;
glutathione-dependent reduction of H
2
O
2
and hydroxyethyl
disulfide by Plrx is negligible. Furthermore, plasmoredoxin
provides electrons for ribonucleotide reductase, the enzyme
catalyzing the first step of DNA synthesis. As demonstrated
by Western blotting, the protein is present in blood-stage
forms of malarial parasites.
Based on these results, plasmoredoxin offers the oppor-
tunity to improve diagnostic tools based on PCR or
immunological reactions. It may also represent a specific
target for antimalarial drug development and is of phylo-

genetic interest.
Keywords: antioxidant; malaria; Plasmodium falciparum;
redox-metabolism; thioredoxin superfamily.
The malarial parasite, Plasmodium falciparum is respon-
sible for more than 2 million deaths per year and novel
antiparasitic drugs are urgently and continuously required
[1,2]. Malarial parasites are exposed to high fluxes of
reactive oxygen species (ROS) and for this reason, proteins
involved in antioxidant defense are promising targets for
antimalarial drug development [3–6]. P. falciparum has been
shown recently to possess two major functional redox
systems: a thioredoxin system [7,8] comprising NADPH,
thioredoxin reductase (TrxR), thioredoxin (Trx) [8,9] and
thioredoxin dependent peroxidases (TPx) [10–14] and a
glutathione system comprising NADPH, glutathione
reductase (GR) [15], glutathione, glutathione S-transferase
[16] and glutaredoxin (Grx) [17].
The thioredoxin superfamily includes the redox-active
proteins thioredoxin, glutaredoxin, tryparedoxin, protein
disulfide isomerase and DsbA (disulfide bond forming
proteins of bacteria) [18,19]. All members of this family
share the Ôthioredoxin-foldÕ consisting of a central five-
stranded b-sheet surrounded by four a-helices [20], and an
active site with two conserved cysteine residues that specify
the biological activity of the protein [18,19]. Thioredoxins
are a group of small ( 12 kDa) proteins with the classical
active site sequence, CGPC. They contribute to a range of
essential cellular functions including protection from ROS,
reduction of enzymes such as ribonucleotide reductase and
thioredoxin peroxidase, and regulation of transcription

factors [18–21]. Mammalian Trx have been shown to
function as cellular growth factors, to modulate apoptosis
and to be highly expressed and secreted by certain tumor
cells [22].
Glutaredoxins with a similar size are part of the
glutathione system and characterized by the active site
sequence, CPYC. They also protect against oxidative
damage, serve as hydrogen-donors for ribonucleotide
reductase and are associated with transcriptional control
[17,19,21,23]. As shown for yeast cells, at least one out of
Correspondence to K. Becker, Interdisciplinary Research Center,
Heinrich-Buff-Ring 26–32, Justus-Liebig-University,
D-35392 Gießen, Germany,
Fax: + 49 641 9939129; Tel.: + 49 641 9939120.
E-mail:
Abbreviations: BSA, bovine serum albumin; GR, glutathione
reductase; Grx, glutaredoxin; GSH, glutathione, reduced;
GSSG, glutathione, oxidized; GST, glutathione S-transferase;
Plrx, plasmoredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase;
TPx, thioredoxin dependent peroxidase.
Note: K.B. and S.M.K. contributed equally to this work.
(Received 2 December 2002, revised 28 January 2003,
accepted 3 February 2003)
Eur. J. Biochem. 270, 1057–1064 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03495.x
four Trx and Grx genes has to be present for viability [24].
The presence of both thioredoxins and glutaredoxins in
different organisms, together with the conservation of their
active sites through evolution, point to the importance of
these antioxidative and regulatory proteins for central
cellular functions. As a third family of redox-active proteins

with functions comparable to Trx and Grx, tryparedoxins
have been described in trypanosomes and crithidiae,
unicellular parasites lacking a glutathione system [25,26].
Here we describe the identification and characterization
of a novel functional redox-active protein in the malarial
parasite, P. falciparum. Together with thioredoxins, gluta-
redoxins and tryparedoxins, this protein represents a
member of the thioredoxin superfamily. The presence of
the protein is restricted to malarial parasites where it is likely
to be involved in ribonucleotide reduction and glutathione
homeostasis.
Materials and methods
PCR
Perfect match primers (forward: 5¢-ATGGCGTGCC
AAGTTGATAA-3¢; reverse: 5¢-TGCTGTCTGTAACCA
CACA-3¢) were designed and PCR was carried out with a
P. falciparum gametocyte cDNA as a template; the forward
primer introduced a BamHI restriction site, the reverse
primer a PstI restriction site. The PCR conditions were
chosen as follows: (a) 94 °C, 30 sec; (b) 80 °C, hold; (c)
94 °C, 30 sec; (d) 60 °C, 30 sec; (e) 72 °C, 2 min; (f)
30 · steps c–e; (g) 72 °C, 3 min; (h) 15 °C, hold. The
amplified 570 bp PCR product was digested with the
corresponding restriction enzymes, purified and cloned into
the expression vector, pQE30, that had been cleaved
previously with BamHI/PstI. The resulting plasmid-con-
struct was sequenced and showed 100% identity to the
genome sequence.
Overexpression and purification of PfPlrx
The Qiagen expression-system (pQE30 vector, that adds an

N-terminal hexahistidyl-tag to the protein for affinity-
purification, and M15 E. coli expression cells) was used for
overexpression and purification of Plrx. The relative mole-
cular mass of the pure protein (as judged by silver stained
SDS/PAGE and gel filtration using a calibrated Sepha-
dex G-75 column) was 21.4 kDa (calculated 21 684 Da).
The calculated absorption coefficient, e
280nm
,ofPfPlrxwas
determined to be 31.4 m
M
)1
Æcm
)1
.
Immunoblotting
Intraerythrocytic stages of P. falciparum were cultured
in vitro as described previously [17]. Rabbit antiserum
raised against recombinant PfPlrx was obtained from
BioScience, Go
¨
ttingen, Germany. The reaction of the
antibodies with authentic PfPlrx in P. falciparum tropho-
zoite extracts, as well as with recombinant protein, was
studied by Western blotting. Samples were subjected to 12%
SDS/PAGE and then blotted on a polyvinylidene difluoride
membrane using a semi-dry blot procedure (50 mA for
55 min). As a secondary antibody, peroxidase-conjugated
porcine anti-(rabbit Ig) Igs (Dako Diagnostika, Hamburg,
Germany) were used.

Enzyme assays
Ribonucleotide reductase activity was determined from the
rate of conversion of [
3
H]GDP into [
3
H]dGDP essentially as
described for CDP reduction [27]. The assay mixture
(200 lL) contained 50 m
M
Hepes, 100 m
M
KCl, 6.4 m
M
MgCl
2
, 500 l
M
GDP (including 1.25 lCi [
3
H]GDP),
100 l
M
dTTP, variable concentrations of PfPlrx, E. coli
thioredoxin, and Trypanosoma brucei thioredoxin, respect-
ively. T. brucei R1 subunit (1 mU, 1.48 l
M
)witha67-fold
molar excess of the R2 subunit (99 l
M

)wasused(1U
corresponds to 1 lmol dGDP formation per min
)1
). The
mixture was incubated at 37 °C for 20 min and the reaction
was stopped by boiling for 10 min. Precipitated protein was
removed by centrifugation at 13 000 g, and products and
educts were dephosphorylated by 45 min incubation with
10 U alkaline phosphatase. Nucleoside, deoxynucleoside
and free bases were then separated isocratically by HPLC
on an Aminex A9 anion exchange column (250 · 4mm)in
100 m
M
sodium borate, pH 8.3 [28,29].
Glutathione reductase [15], thioredoxin reductase [9] and
trypanothione reductase activities [29,30] were determined
spectrophotometrically at 340 nm monitoring the consump-
tion of NADPH as described previously. In these assays up
to 100 l
M
PfPlrx was tested as the substrate. The detection
limit in these assays is DA ¼ 0.002Æmin
)1
, that corresponds
to an NADPH oxidation rate of 0.3 l
M
Æmin
)1
and an
activity of 0.3 mUÆmL

)1
of an NADPH dependent disulfide
reductase. The insulin-reduction assay is described in the
legend to Fig. 3; P. falciparum thioredoxin used for this
assay was expressed and purified as described previously [9].
Reaction of PfPlrx with different reducing agents
Reduction of PlrxS
2
by trypanothione and tryparedoxin.
In trypanosomes and other Kinetoplastida, a major relay
system of electron transferring reactions exists that
comprises of NADPH, trypanothione reductase, trypano-
thione, tryparedoxin and a terminal acceptor such as
ribonucleotide reductase [30]. The interactions of Plrx with
this system were tested as described for the GHOST assay [7].
Briefly, 1 mL assay mixture at 25 °Cwereused.The
compounds were added in the following order: buffer
(40 m
M
Hepes, 1 m
M
EDTA at pH 7.5), NADPH (200 l
M
final concentration), Trypanosoma cruzi trypanothione
reductase (80 n
M
¼ 0.25 enzyme units), PlrxS
2
, trypanothi-
one disulfide (20–50 l

M
)andT. brucei tryparedoxin disul-
fide (4.5 l
M
). In a series of assays, the order of additions was
changed so that PlrxS
2
was added at differing steps in the
sequence of additions.
In the course of the reaction sequence, essentially, each
disulfide is reduced completely to the corresponding dithiol,
NADPH oxidation being the driving force. After each
addition to the assay mixture, the absorbance decrease at
340 nm due to NADPH oxidation was registered and
the rate of the respective reaction was calculated according
to the equation: v ¼ Dc · min
)1
¼ DA/(1 min · e · 1cm)
[lM · min
)1
], where the e)value for NADPH is
6.22 m
M
)1
Æcm
)1
. From a given value of v, the rate constant
1058 K. Becker et al. (Eur. J. Biochem. 270) Ó FEBS 2003
k was determined using the equation for a second order
reaction: k ¼ v/{[R(SH)

2
] · [PlrxS
2
]}. Assay conditions for
the reduction of Plrx by other reducing agents are given in
thelegendtoTable1.
Reduction of GSSG by PfPlrx
PfPlrx was prereduced with 1 m
M
dithiothreitol. The
protein was then separated rapidly from excess dithiothre-
itol by affinity chromatography using Ni-nitrilotriacetic acid
agarose. Reduced PfPlrx (12.5 or 25 l
M
) was then incuba-
ted for 30 s and 15 min at 4 °Cand25 °C, respectively, with
GSSG (25 or 50 l
M
)in50m
M
potassium phosphate, 1 m
M
EDTA, 200 m
M
KCl at pH 6.9. This incubation was
followed by addition of 100 l
M
NADPH and 50 mUÆmL
)1
human glutathione reductase in order to determine the

concentration of residual GSSG. PfPlrx was found to reduce
GSSG in a nonenzymatic reaction. The rate constant, k,
of this reaction was calculated as v/[Plrx(SH)
2
] ·
[GSSG] · min on the basis of the following experiment.
Reduction of 25 l
M
GSSG (25 °C for 30 s) with PfPlrx
(12.5 l
M
)ledto19l
M
residual GSSG; this corresponds to
the reduction of 12 l
M
GSSG per min. Thus, k was
calculated to be 0.0384 l
M
)1
Æmin
)1
. In parallel experiments,
we removed Plrx after the reaction with GSSG using
Ni-nitrilotriacetic acid agarose. Subsequently, the thiol
content, representing the formed GSH, was measured in
the solution.
Results and discussion
In the genome of the malarial parasite, P. falciparum [31] a
gene showing sequence similarities with thioredoxin genes

was identified. The sequence consisted of an exon contain-
ing 537 bp located on chromosome 3. The gene was
amplified by PCR using a gametocyte cDNA as a template,
sequenced, cloned into an expression vector, and over-
expressed in E. coli. The deduced amino acid sequence
(PfPlrx; accession number AAF87222) comprised 179
residues (22 kDa) and contained the unique active site
motif, WCKYC, when compared with other members of
the thioredoxin superfamily. The novel protein was named
plasmoredoxin (Plrx). Putative plasmoredoxins of compar-
able size were also identified by in silico analyses in the
genomes of the Plasmodium species, P. vivax [32], P. berg-
hei, P. yoelii,andP. knowlesi (this paper). The correspond-
ing amino acid sequence alignments showed identities of
67.4, 66.9, 72.6 and 67.2% with P. falciparum plasmo-
redoxin (Fig. 1). The identity of PfPlrx with other members
of the thioredoxin superfamily, for example PfTrx (31.4%)
or PfGrx (27.5%) were significantly lower. Apart from
members of this superfamily, the highest degrees of identity
(31.3 and 32.6%) were with ResA (P35160), a respiration
regulating protein of Bacillus subtilis, and HelX (M96013),
a putative periplasmic disulfide oxidoreductase of the
photosynthetic bacterium, Rhodobacter capsulatus, respect-
ively. Homology modelling based on the
SWISS PROT
program resulted in a partial three-dimensional structure
of Plrx. Residues 43–94, representing 28% of the complete
amino acid sequence were modelled and indicated a
characteristic thioredoxin fold including the active site
sequence, WCKYC. In a reconstructed phylogenetic tree,

plasmoredoxins cluster as one group separate from thio-
redoxins, glutaredoxins and tryparedoxins (Fig. 2). Within
the plasmoredoxins, the rodent parasites P. yoelii and
P. berghei Plrx share the highest degree of amino acid
identity (91%), followed by the P. vivax/P. knowlesi pair
with 87.6%. P. falciparum are in between these two groups.
This result suggests a close relationship between P. knowlesi
that infects monkeys and P. vivax that causes tertian
malaria in man.
Interestingly, two similar sequence annotations (Gen-
Bank accesson numbers, NP_473166 and CAB38989) were
available that proposed a large protein of 2417 and 2396
amino acids, respectively, with a putative structural function
in the cytoskeleton of P. falciparum. These annotations
suggested that plasmoredoxin might be part of this large
protein as a possible second exon. To check this possibility,
a PCR with Ôexon-overlappingÕ primers [one primer in
putative exon 1 (the big structural protein), the other primer
Table 1. Reduction of PlrxS
2
by dithiols and glutathione at 25 °C.
Reductant k · 10
3
[l
M
)1
Æmin
)1
] k [
M

)1
Æs
)1
] Conditions
NADPH £ 0.01
Dithiothreitol 1.4 23 pH 8.0, 100 m
M
Tris
Dihydrolipoamide 2.0 33 pH 7.4, 50 m
M
phosphate, 1 m
M
EDTA
GSH
a
0.28 4.7 pH 8.0, 100 m
M
Tris
0.10 1.6 pH 7.4, 100 m
M
Tris
0.03 0.5 pH 6.9, 100 m
M
Tris
P. falciparum glutaredoxin
b
14 230 pH 7.4, 100 m
M
Tris
P. falciparum thioredoxin

c
2.2 37 pH 7.4, 100 m
M
phosphate
Trypanothione
d
0.67 11 pH 7.5, 40 m
M
Hepes
T. brucei brucei tryparedoxin
d
30 503 pH 7.5, 40 m
M
Hepes
a
Assays were performed in 100 m
M
Tris, 1 m
M
EDTA, at different pH values adjusted at 25 °C, in the presence of 200 l
M
NADPH,
1UÆmL
)1
PfGR, 0.5–10 m
M
GSH, and 50 l
M
PlrxS
2

. For the reaction of PlrxS
2
with glutathione, the limited data set did not allow us to
distinguish between pseudosecond and third order kinetics.
b
Assays were performed as above but in the presence of 1 m
M
GSH and
10–60 l
M
PfGrx. At Grx concentrations ‡ 20 l
M
no clear increase in DAÆmin
)1
value was detected. The rate constant was therefore
calculated on the basis of the value determined for 10 l
M
Grx.
c
The reaction of Plrx (25–50 l
M
) with thioredoxin (100 l
M
) was determined
in 100 m
M
potassium phosphate, 2 m
M
EDTA, pH 7.4 in the presence of 1 UÆmL
)1

PfTrxR and 200 l
M
NADPH.
d
Assay conditions for
the reaction of Plrx with the trypanothione system are given in the Materials and methods section.
Ó FEBS 2003 Malarial plasmoredoxin, a novel redox-active protein (Eur. J. Biochem. 270) 1059
in putative exon 2 (the plasmoredoxin)] was performed
using PfcDNA as a template. Under various PCR condi-
tions, however, no product was obtained indicating that
PfPlrx is unlikely to represent a part of the protein encoded
by exon 1 and indeed, very recently both former sequence
predictions were updated and split into two parts resulting
in a putative protein of 2226 amino acids and a second
predicted protein of 179 amino acids representing plasmo-
redoxin.
As summarized in Table 1, PfPlrx can be reduced by
different dithiols as well as by GSH. Most effective were
P. falciparum glutaredoxin and T. brucei tryparedoxin.
Fig. 1. Alignment of the amino acid sequence of plasmoredoxin from Plasmodium falciparum with putative homologues of different Plasmodium species.
Pf, P. falciparum (GenBank AAF87222); Pv, P. vivax (GenBank AAF99466); Py, P. yoelii (GenBank EAA16465; gnl|py|TIGR_c5m141); Pk,
P. knowlesi (gnl|pk|Sanger_PKN.0.004551); Pb, P. berghei (gnl|pbgss|UFL_249PbC01, gnl|pbgss|UFL_204PbH08, gnl|pbgss|UFL_225PbD05),
this sequence was generated from three different genomic clones and is likely to lack a small fragment of the sequence. Identical amino acids are
highlighted, the putative active site is boxed.
Fig. 2. Phylogenetic relations of plasmoredoxins, thioredoxins, glutaredoxins, and tryparedoxins. Plasmoredoxins represent a novel family of redox-
active proteins belonging to the thioredoxin superfamily. The sequence comparisons were carried out using the
CLUSTAL W
program of the EMBL
European Bioinformatics Institute (www2.ebi.ac.uk./clustalW/). Pk, P. knowlesi;Pv,P. vivax;Pf,P. falciparum;Py,P. yoelii;Pb,P. berghei;Hs,
Homo sapiens;Ec,E. coli;Tb,T. brucei;Cf,Crithidia fasciculata;Tc,T. cruzi; Plrx, plasmoredoxin; Trx, thioredoxin; Trp, tryparedoxin; Grx,

glutaredoxin.
1060 K. Becker et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Whether the reduction of Plrx by GSH is physiologically
significant might be questioned as the pseudosecond order
rate constant was only 1.6
M
)1
Æs
)1
at pH 7.4 and 25 °C.
Concentration-dependent redox activity of Plrx was dem-
onstrated by its ability to cleave disulfide bonds of insulin
when using dithiothreitol as a source of reducing equivalents
(Fig. 3). In this assay, P. falciparum thioredoxin served as a
positive control and dithiothreitol as well as bovine serum
albumin as negative controls. Using the glutathione system
as a primary source of reducing equivalents, the insulin-
reduction by 5 l
M
Plrx was too slow to be detected at the
physiological pH of 7.4 but a clear reaction was apparent at
pH 8.0.
Interestingly, PfPlrx was found to be no substrate for
thioredoxin reductase from P. falciparum, E. coli, and man;
of glutathione reductase from P. falciparum and man and
trypanothione reductase from T. cruzi.Ineachcase,the
specific activity was below the detection limit of
25 mUÆmg
)1
enzyme protein.

To test whether Plrx modulates glutathione reductase and
thioredoxin reductase activity, respectively, Plrx was pre-
reduced by incubation with 2 m
M
dithiothreitol. Residual
dithiothreitol was removed by affinity chromatography on a
Ni-nitrilotriacetic acid column. Directly after elution, 20 l
M
Plrx(SH)
2
was added to a standard GR assay, pH 6.9 [15],
and a TrxR assay, pH 7.4, containing 20 l
M
PfTrx [9],
respectively. The addition of reduced Plrx did not influence
the reaction catalysed by the disulfide reductases at 25 °C.
The ability of PfPlrx to reduce hydroxyethyl disulfide
GSH-dependently was tested in an assay system typically
used for characterizing glutaredoxins [17]. The assay (in
100 m
M
Tris, 1 m
M
EDTA, pH 8.0) contained 100 l
M
NADPH, 0.25 UÆmL
)1
PfGR, 1 m
M
GSH as well as

different concentrations of PfGrx and PfPlrx, and was
started with 735 l
M
hydroxyethyl disulfide. In a reference
cuvette containing no Grx/Plrx, the spontaneous reaction
between GSH and hydroxyethyl disulfide was accounted
for. Grx (20 n
M
) produced an DAÆmin
)1
value of 0.051,
corresponding to a k
cat
of 410 min
)1
(see also [17]). Plrx (25
and 75 l
M
)resultedinDAÆmin
)1
values of 0.025 and 0.070,
respectively, corresponding to a k
cat
of 0.15 min
)1
. Thus, the
GSH-dependent hydroxyethyl disulfide reducing activity of
PfPlrx is by a factor of almost 3000 lower than the activity of
PfGrx1 [17].
Peroxidase activity of PfPlrx was tested in 100 m

M
Tris,
1m
M
EDTA, pH 7.4 (or 8.0) in the presence of 200 l
M
NADPH, 1 UÆmL
)1
PfGR, 2 m
M
GSH and 50 l
M
PlrxS
2
.
After 15 min preincubation, which guaranteed the reduc-
tion of PlrxS
2
,200l
M
H
2
O
2
was added. The resulting
DAÆmin
)1
value was higher by £ 0.01 than the one of the
controls carried out in the absence of Plrx at both pH
values. This indicated an extremely slow reaction between

Plrx(SH)
2
and H
2
O
2
– the second order rate constant being
£ 1.6 · 10
)4
lM
)1
Æmin
)1
– when comparing Plrx with
known peroxidases of P. falciparum [10–14].
Plasmoredoxin, in its dithiothreitol-reduced form, was
tested successfully as a hydrogen donor for T. brucei
ribonucleotide reductase. This result points to an in vivo
contribution of PfPlrx to DNA synthesis. The reduction of
ribonucleotide reductase is, in most organisms, produced by
Trx and Grx; in Trypanosomes, tryparedoxin was shown to
have a comparable function [18,19,25,29].
Reduced PfPlrx was furthermore shown to reduce
quantitatively glutathione disulfide. A 15-min incubation
of 25 l
M
PfPlrx with 50 l
M
GSSG resulted in the formation
of 50 l

M
GSH, as indicated by a decrease of the GSSG
concentration from 50)25 l
M
. The concomitant determi-
nation of 44.2 l
M
GSH makes it unlikely that glutathion-
ylated Plrx is a major reaction product. The following
reaction scheme is therefore proposed:
PfPlrxðSHÞ
2
þ GSSG ! PfPlrx ðS-SÞþ2 GSH
According to the data obtained with different substrate
concentrations and incubation times, the lower limit of the
k-value for this chemical reaction can be estimated as
0.01 l
M
)1
Æmin
)1
at 4 °C and of 0.04 l
M
)1
Æmin
)1
at 25 °C.
For many thioredoxins (with the notable exception of
PfTrx) the corresponding rate constant is £ 0.01 l
M

)1
Æmin
)1
at 25 °C[9].
The reduction of GSSG is, in most organisms, conducted
by the NADPH-dependent flavoenzyme, glutathione
reductase (GR) [3]. However, we have shown recently that
insects including Drosophila melanogaster and Anophe-
les gambiae lack a genuine GR although they contain high
concentrations of glutathione [33]. In this context, a
nonenzymatic reduction of GSSG by reduced thioredoxin
was described for different organisms and proposed to have
Fig. 3. Insulin-reduction activity of PfPlrx in comparison with PfTrx. In
this assay, the precipitation of reduced insulin B-chains is followed at
600 nm. One ml of reaction mixture contained 0.17 m
M
porcine insulin
in 50 m
M
Tris/HCl, 2 m
M
EDTA at pH 7.4. The reaction was started
at 25 °C by adding 1 m
M
dithiothreitol in the presence of 2 l
M
PfTrx
(closed square), 2 l
M
PfPlrx (closed triangle) or 5 l

M
PfPlrx (cross).
1m
M
dithiothreitol without protein (closed diamond) served as a
negative control. Addition of 5 l
M
bovine serum albumin to the
dithiothreitol control gave identical results. In additional assays,
dithiothreitol was replaced by a reducing system consisting of either
200 l
M
NADPH, 1 UÆmL
)1
PfGR, 2 or 10 m
M
GSH, 5 l
M
Plrx or of
200 l
M
NADPH, 1 UÆmL
)1
PfTrxR, 5 l
M
Plrx at pH 6.9, 7.4 and 8.0.
Only at pH 8.0 and 10 m
M
GSH was a clear insulin reducing activity
observed within 30 min (open square). At pH 8.0 the reduction of

5 l
M
Plrx by 1 m
M
dithiothreitol (closed circle) was also more efficient
than at pH 7.4.
Ó FEBS 2003 Malarial plasmoredoxin, a novel redox-active protein (Eur. J. Biochem. 270) 1061
in vivo relevance [9,33]. Obviously, PfPlrx, as a member of
the thioredoxin superfamily, is also able to fulfil this
function. The stoichiometric reaction observed for PfPlrx
and GSSG may contribute to antioxidant defense and
specific redox regulatory processes in malarial parasites that
grow and multiply in an environment of high oxygen
tension [34].
P. falciparum plasmoredoxin is a member of a novel
family of redox active proteins belonging to the thioredoxin
superfamily. PfPlrx is larger than classical thioredoxins,
glutaredoxins and tryparedoxins, it shares, however, typical
structural and functional characteristics with the other three
groups. The reactions of P. falciparum plasmoredoxin with
rabbit IgG raised against the recombinant protein were
demonstrated by Western blotting. As shown in Fig. 4,
single bands of expected sizes (24 kDa, due to the His-tag
and 22 kDa) appeared when probing the recombinant
protein and a trophozoite extract of P. falciparum.This
result, and the fact that PfPlrx was amplified from a cDNA
library, indicate that the gene is transcribed and the protein
is present in blood-stage forms of the parasite that cause
malaria in the human host. Considering the above may
allow a unique avenue for developing diagnostic tools based

on PCR or immunological methods. Many organisms,
including E. coli, D. melanogaster, yeast and man possess
more than one Trx or Grx. As indicated by our studies,
P. falciparum possesses at least one thioredoxin [9], at least
two glutaredoxin-like proteins [8,17] and the newly discov-
ered plasmoredoxin. In this context it is furthermore
interesting to note that until now the gene of only one
glutathione S-transferase has been detected in the genome
of P. falciparum [16] and that no glutathione-dependent
peroxidase has been discovered so far. The gene believed to
represent a GPx, was found to code for a thioredoxin
dependent peroxidase [12]. Taken together, these data might
indicate that P. falciparum does not use glutathione
dependent reactions to the extent described for other
organisms. In other words, malarial parasites might have
developed a unique additional defense line against oxidative
stress, and an additional source of reducing equivalents for
deoxyribonucleotide synthesis as well as for signalling
processes. Indeed, the multiplication rate of P. falciparum
is among the fastest in eukaryotic organisms. Potential roles
of plasmoredoxin in redox metabolism of P. falciparum are
delineated in Fig. 5.
Fig. 4. Western blot of P. falciparum plasmoredoxin. Lane 1: Recom-
binantly produced P. falciparum plasmoredoxin (200 ng); lane 2:
extractoftheP. falciparum strain 3D7 (18 lg total protein). The
molecular masses of the standard proteins on lane 3 are given on the
right hand side.
Fig. 5. The putative roles of P. falciparum
plasmoredoxin (Plrx) in redox metabolism of
the parasite. Only proteins/pathways that have

beenverifiedtoexistinP. falciparum are
shown. NADPH represents the major source
of reducing equivalents in the infected
erythrocyte. Both, thioredoxin reductase
(TrxR) and glutathione reductase (GR) reduce
their respective substrates, thioredoxin (Trx)
and glutathione disulfide (GSSG) by using
NADPH. Trx reduces Trx-dependent peroxi-
dases as well as ribonucleotide reductase
(RiboR). Reduced glutathione (GSH) serves
as a substrate of glutathione S-transferase
(GST) or reduces glutaredoxin, which in turn
is able to provide RiboR with electrons.
Plasmoredoxin is, like thioredoxin, able to
reduce RiboR as well as GSSG and can be
reduced by GSH, Trx, and glutaredoxin.
1062 K. Becker et al. (Eur. J. Biochem. 270) Ó FEBS 2003
According to our data, Plrx seems to be present in
Plasmodium species only, rendering the protein and/or the
gene a specific diagnostic tool for clinical and epidemiolo-
gical studies. Furthermore, Plrx as an antioxidant protein
that is also involved in DNA synthesis may represent a
potential drug target.
Acknowledgements
The technical assistance of Elisabeth Fischer, Petra Harwaldt and Beate
Hecker is acknowledged. The authors wish to thank R. L. Krauth-
Siegel, Heidelberg, for performing the ribonucleotide reductase assays
and for kindly providing the components of the tryparedoxin-reducing
system. We also thank Julia K. Ulschmid and Scott Mulrooney for
helpful discussion. Sequence data for P. vivax/berghei/falciparum was

obtained from the University of Florida Gene Sequence Tag Project
Website at: med.ufl.edu. Funding was provided by
the National Institute of Allergy and Infectious Diseases (for P. berghei
and P. vivax data) and University of Florida Division of Sponsored
Research and the Burroughs Wellcome Fund (for P. falciparum data).
Sequence data for P. falciparum chromosome 3 was obtained from The
Sanger Centre website at />rum/. Sequencing of P. falciparum chromosome 3 was accomplished as
part of the Malaria Genome Project with support by The Wellcome
Trust. Our work on redox metabolism of malarial parasites is
supported by the Deutsche Forschungsgemeinschaft (grants, SFB 535
to K. B. and Schi 102/8-1 to R. H. S.).
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