The Putative Glutathione Peroxidase Gene of Plasmodium
falciparum Codes for a Thioredoxin Peroxidase*
Helena
Sztajer
,
Benoit
Gamain§,
Klaus-Dieter
Aumann¶,
Christian
Slomianny
,
Katja
Becker**,
Regina
Brigelius-Flohé
, and
Leopold
Flohé
§§
From the
Department of Biochemistry, Technical
University of Braunschweig, Mascheroder Weg 1, 38124 Braunschweig,
Germany, § NIAID, National Institutes of Health, Bethesda,
Maryland 20892, ¶ National Centre for Biotechnology, Mascheroder
Weg 1, 38124 Braunschweig, Germany,
University of Science and
Technology Lille, INSERM EPI 9938, Batiment SN3, 59655 Villeneuve
d'Ascq Cedex-France, ** Research Center for Infectious Diseases,
Würzburg University, Röntgenring 11, 97070 Würzburg,
Germany, and 
German Institute of Human
Nutrition, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany
Received for publication, September 20, 2000, and in revised form, October 27, 2000
 |
ABSTRACT |
A putative glutathione peroxidase gene
(Swiss-Prot accession number Z 68200) of Plasmodium
falciparum, the causative agent of tropical malaria, was
expressed in Escherichia coli and purified to
electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was
active with H2O2 and organic hydroperoxides
but, unlike phospholipid hydroperoxide glutathione peroxidase, not with
phosphatidylcholine hydroperoxide. With glutathione peroxidases it
shares the ping-pong mechanism with infinite
Vmax and Km when analyzed
with GSH as substrate. As a homologue with selenocysteine replaced by
cysteine, its reactions with hydroperoxides and GSH are 3 orders of
magnitude slower than those of the selenoperoxidases. Unexpectedly, the
plasmodial enzyme proved to react faster with thioredoxins than with
GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as
thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also
displays ping-pong kinetics, yet with a limiting Km of 10 µM and a k1' of 0.55 s
1. The apparent k1'
for oxidation with cumene, t-butyl, and hydrogen peroxides
are 2.0 × 104
M
1 s
1,
3.3 × 103 M
1
s
1, and 2.5 × 103 M
1 s
1,
respectively. k2' for reduction by autologous
thioredoxin is 5.4 × 104 M-1
s
1 (21.2 M
1 s
1
for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.
 |
INTRODUCTION |
Plasmodium falciparum, the causative agent of the most
severe form of malaria, reportedly displays glutathione peroxidase (GPx)1 activity (1). Also, a
putative GPx gene was isolated from P. falciparum (2).
Complementing enzymes that may constitute a
glutathione-dependent antioxidant defense system have also
been characterized in Plasmodia species. They can rely on
their own GSH biosynthesis (3), and the pertinent key enzyme,
-glutamylcysteine synthetase, was identified (4). The parasites
regenerate GSH from GSSG more efficiently than their host cells (3),
and a plasmodial glutathione reductase was also characterized (5). Plasmodia species require an efficient antioxidant defense
system, since they have to survive in a pro-oxidant habitat, the red
blood cell. Moreover, they have to overcome the oxidant attack of
phagocytes during the critical period between dissemination from, and
reinvasion of the host cell (6, 7). Within the infected erythrocytes, Plasmodia species appear to enhance oxidative stress, as
indicated by the generation of methemoglobin (8) and hydroxyalkenals (9, 10). On the other hand, they are known to be sensitive to oxidant
killing, as evident from the peroxide nature and pro-oxidant potential
of many antimalarial drugs and impaired survival in host cells with
disturbed hydroperoxide metabolism (for review see Ref. 11). Plasmodial
enzymes involved in the antioxidant defense of the parasite have
therefore attracted attention as potential targets for the development
of novel antimalarials.
In this context the putative plasmodial GPx gene appeared particularly
intriguing. Encoding a sulfur homologue of the mammalian selenoproteins, the gene product should be substantially less efficient
than the host cell enzymes. (12, 13). The comparatively high
sensitivity of Plasmodia species to hydroperoxides could therefore result from the necessity to rely on sulfur-catalyzed hydroperoxide reduction, whereas the host cells make use of the more
efficient selenium catalysis. Beyond, the amino acid sequence deduced
from the plasmodial GPx gene resembles phospholipid hydroperoxide peroxidase (PHGPx, GPx-4), which displays a degenerate substrate specificity (14). Correspondingly, it could not be taken for granted
that the plasmodial GPx homologue is indeed a glutathione peroxidase.
The plasmodial GPx gene was therefore heterologously expressed in
Escherichia coli, and the protein was purified in sufficient
quantities to allow an in-depth functional analysis. As expected, it
proved to be a peroxidase acting on a broad spectrum of hydroperoxides
with low efficiency. Also, the reaction rates with GSH were
surprisingly low. Instead, thioredoxins reduced the enzyme efficiently
enough to reclassify this member of the glutathione peroxidase family
as a thioredoxin peroxidase and to substitute the acronym PfTPx for the
originally introduced glutathione peroxidase of P. falciparum (PfGPx) (2).
 |
EXPERIMENTAL PROCEDURES |
Heterologous Expression and Purification of PfTPx--
The
full-length cDNA encoding the PfTPx was amplified by reverse
transcriptase-polymerase chain reaction using asynchronous blood stage
RNA and cloned into the pET5a vector (Calbiochem-Novabiochem) between
the EcoRI and NdeI sites and transformed into
E. coli BL21 (DE3) pLysS.
The clone was grown in carbenicillin (1 mg/ml)-supplemented LB media at
37 °C and 180 rpm to an A600 of 0.5 and
induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside. The culture was grown for an additional 5 h and controlled for expression by
SDS-polyacrylamide gel electrophoresis. For routine preparation, cells
were harvested 3 h after induction, resuspended in 50 mM Tris, 1 mM dithiothreitol, pH 8.5 (buffer
A), disrupted with a French press at the 900 p.s.i., and
centrifuged at 18,000 rpm for 10 min. The supernatant was loaded on a
Macro-Prep® High Q Support fitted on a
Macro-Prep® High S Support column with a flow rate of 2 ml/min (Bio-Rad). The column was washed with 10 bed volumes of buffer
A. The Macro Prep® High S Support column alone was eluted
with a NaCl gradient (0-2 M) in the same buffer. Fractions
with GPx activity were concentrated by ultrafiltration (Omega Cell,
cut-off 10 kDa, Pall Gelman Sciences, An Arbor, MI), loaded onto a
Sephacryl S-200 (Amersham Pharmacia Biotech) gel filtration column
equilibrated with 0.1 M Tris, 0.1 M NaCl, 5 mM EDTA, pH 7.6, and eluted at a flow rate of 0.5 ml/min. Active fractions were analyzed by SDS-polyacrylamide gel
electrophoresis for homogeneity and stored at 4 °C.
Characterization of Expression Product--
The molecular mass
of denatured PfTPx was estimated by silver-stained (15)
SDS-polyacrylamide gel electrophoresis using the Phast
SystemTM (Amersham Pharmacia Biotech) with an acrylamide
gradient of 8-25% and a 10-kDa ladder as reference. The molecular
mass of native PfTPx was estimated by gel filtration on a Sephadex
S-100 column equilibrated with a 0.1 M Tris buffer of pH
7.6 containing 5 mM EDTA at a flow rate of 0.5 ml/min.
Chymotrypsinogen, bovine serum albumin, blue dextran, and cytochrome
c were co-chromatographed as reference proteins. Protein
concentration was determined according to Bradford (16) with the
reagent from Bio-Rad, taking bovine serum albumin as the standard. The
precise molecular weight was determined by matrix-assisted laser
desorption and ionization time of flight (MALDI-TOF) mass spectrometry
with a Bruker Reflex II-MALDI-TOF mass spectrometer
(Bruker-Franzen-Analytik, Bremen, Germany). For this purpose the
protein was precipitated with trichloroacetic acid, washed with the
acetone, and resuspended in saturated matrix solution (10 mg/ml
sinapinic acid in 40% acetonitrile and 0.1% trifluoroacetic acid).
The protein at a final concentration about 50 pmol/µl matrix solution
was accelerated at 20 kV. Spectra were externally calibrated using
bovine serum albumin as standard. Approximately 200 shots were summed
for each spectrum. N-terminal sequencing was performed with an Applied
Biosystems 494 A sequencer.
Activity Measurement and Kinetic Analysis--
GPx activity was
measured by monitoring the glutathione reductase-catalyzed NADPH
oxidation at 340 nm at 30 °C in 0.5 ml containing 0.3 mM
NADPH in 0.1 M Tris, pH 7.6, 0.1% Triton, 5 mM
EDTA, 3.3 mM GSH, and 73 µM
t-butyl hydroperoxide (t-bOOH) (17). Specificity for the
hydroperoxide substrate was investigated with t-bOOH (Merck), cumene
hydroperoxide (Merck Eurolab, Darmstad, Germany),
H2O2 (Sigma-Aldrich), and phosphatidylcholine
hydroperoxide prepared according to Maiorino et al. (18).
The concentration of hydroperoxides was determined by allowing the GPx
reaction to run to completion. For quantification of t-bOOH,
H2O2, and cumene hydroperoxide bovine GPx
(Sigma-Aldrich), for phosphatidylcholine hydroperoxide a PHGPx preparation of rat testis (19) was used.
Thioredoxin peroxidase activity was determined analogously by replacing
GSH by thioredoxin and glutathione reductase by thioredoxin reductase.
In each case the particular thioredoxin was coupled to the thioredoxin
reductase of the same species. Care was taken that the thioredoxin
reductase capacity never became rate-limiting. E. coli
thioredoxin was from Sigma-Aldrich, human thioredoxin was kindly
provided by Prof. A. Holmgren Uppsala, Sweden, and thioredoxin as well
as thioredoxin reductase of P. falciparum were prepared as
described elsewhere (20, 21).
For kinetic analysis, the glutathione peroxidase assay was performed at
GSH concentrations ranging from 3.3 to 30 mM and various hydroperoxides (t-bOOH, H2O2, cumene
hydroperoxides). Thioredoxin peroxidase kinetics were performed with
thioredoxin of P. falciparum at concentrations between 1.5 and 5 µM. Spontaneous reaction rates were subtracted.
Kinetic data were obtained by the single curve progression analysis
according to Forstrom et al. (22) and further analyzed as
described by Dalziel (23) for bisubstrate reactions.
 |
RESULTS |
Heterologous Expression and Purification of PfTPx--
E.
coli BL21 (DE3) pLys cells transformed with the pET5a-derived
expression plasmid efficiently produced PfTPx upon induction with
isopropyl-1-thio-
-D-galactopyranoside, as indicated by
the appearance of a prominent band of ~20 kDa. The heterologously expressed protein peaked near 2 h after induction and appeared to
remain stable for at least three more hours. The purification scheme
applied yielded a product of apparent electrophoretic homogeneity (Fig.
1). The apparent as well the precise
molecular mass of 19,750 Da obtained by MALDI-TOF spectrometry was
significantly less than the molecular mass of 23953 Da calculated for
the deduced amino acid sequence of full-length PfTPx (205 residues).
N-terminal sequencing revealed an amino acid sequence covering position
26-40 of the deduced PfTPx sequence shown in Fig.
2. Within experimental error, the
experimentally determined molecular mass complies with that calculated
for a sequence covering position 26 to 196 (19,780.8 Da).

View larger version (110K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of heterologously expressed
PfTPx. Lane 1, total protein of E. coli
lysate, as obtained at harvest (3 h after induction). Lane
2, PfTPx as obtained by the purification scheme applied (see
"Experimental Procedures." Lane 3, molecular mass
standards (10-kDa ladder). Typical yields ranged around 3 mg from a
1-liter culture.
|
|

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 2.
Alignment of PfTPx with four distinct types
of human glutathione peroxidases: PHGPx (Swiss-Prot accession number
P36969), extracellular GPx (pGPx; Swiss-Prot accession number P22352),
gastrointestinal GPx (giGPx; Swiss-Prot accession number P18283), and
cGPx (Swiss-Prot accession number P07203). Amino acid residues of
PfTPx conserved in other GPx types are shadowed. Residues
constituting the catalytic triad (Cys-76, Gln-111, and Trp-169) are
marked with an asterisk. Residues implicated in specific
binding of GSH by cGPx are marked with an arrow.
|
|
Comparison of PfTPx with cGPx and PHGPx--
Alignment of PfTPx
with mammalian glutathione peroxidases reveals that the plasmodial
enzyme is a member of the GPx superfamily yet is more related to PHGPx
than to any of the other GPx types (Fig. 2) displaying 64, 55, 51, and
45 identities with PHGPx, cGPx, gastrointestinal GPx, and pGPx,
respectively. Also the PfTPx sequence as obtained by heterologous
expression in E. coli, corresponds to the homologous stretch
of 170 amino acid residues found in mature porcine PHGPx (24). Like
PHGPx, PfTPx proved to be monomeric when subjected to Sephadex G-100
gel filtration under nondenaturing conditions. The nativity of the
eluting enzyme was verified by GPx activity measurements. It eluted
with an apparent molecular mass of 18.9 kDa, whereas no activity could
be detected in the fractions corresponding to the molecular mass of its
tetrameric congeners, pGPx or cGPx. As is evident from the x-ray
structures of cGPx (25) and pGPx (26), subunit contact surfaces in
these tetrameric GPx types are essentially built up by residues
corresponding to the inserts at positions 121 and 161 of the PfTPx
sequence (27). They are missing in both PfTPx and PHGPx, which explains their monomeric nature.
PfTPx, like PHGPx, was more active with cumene hydroperoxide, less
active with H2O2 and t-bOOH, and unlike PHGPx,
did not at all accept phosphatidylcholine hydroperoxide (data not
shown). In terms of molar efficiencies, PfTPx appeared markedly poorer than bovine cGPx coinvestigated as a selenoperoxidase reference standard. Specific GPx activities, as measured under routine
conditions, differed by three orders of magnitude (not shown).
The kinetic mechanism for GSH-dependent hydroperoxide
reduction by PfTPx was evaluated by means of the single curve
progression analysis (22) at various fixed GSH concentrations, which
were kept constant over time by regeneration, and a suboptimal
concentration of ROOH, which declined over time and correspondingly
lead to slowing down of the reaction rate. From these curves, the
reciprocal concentrations of ROOH at intervals of 2 s were derived
and plotted against the initial velocities at each pertinent time
point, as exemplified for the turnover of t-bOOH by GSH in Fig.
3. The data are presented as Dalziel
plots (23), in which the reciprocal velocities are multiplied by enzyme
molarities to facilitate the extrapolation of meaningful kinetic
coefficients, as indicated. As shown in Fig. 3, such primary plots
yielded parallel slopes for different concentrations of GSH, as is
typical for "enzyme substitution" or "ping-pong" mechanisms.
Replotting the reciprocal GSH concentrations against the reciprocal
apparent Vmax for infinite concentrations of
t-bOOH yielded a straight line cutting at the ordinate origin (Fig.
4). The same kinetic pattern was
displayed by PfTPx with H2O2 and cumene
hydroperoxide as long as GSH was used as reducing substrate (Table
I). It can be described by a Dalziel
equation for two substrate ping-pong mechanisms.
|
(Eq. 1)
|
wherein the coefficient
0 approximates zero.
Accordingly, limiting Vmax and
Km values are infinite. Such lack of enzyme
saturation can be due to two distinct catalytic phenomena, either the
formation of enzyme-substrate complex is slower than the reaction
within the complexes or specific enzyme-substrate complexes not formed
at all, as is presumed in Equations 2-4.
|
(Eq. 2)
|
|
(Eq. 3)
|
|
(Eq. 4)
|
In this case, the coefficient
1 is defined as the
reciprocal rate constant k
for the net forward reaction of reduced enzyme with ROOH and depends on the nature
of the peroxide (Table I). k
is
defined as k+1
k
1,
and may be regarded as k+1, since the partial
reaction shown in Equation 2 should be irreversible.
2
is the reciprocal k
for the two-step
regeneration of the reduced enzyme by GSH according to Equations 3 and
4. Therefore, the physical meaning of k
is more complex (Equation 5).
|
(Eq. 5)
|
The kinetic pattern of PfTPx is identical to that of the
selenium-containing glutathione peroxidases (28-31). However, the kinetic coefficients
1 and
2 of PfTPx
differ markedly from those of the selenoproteins. Instead, they are
similar to those of a sulfur homologue of PHGPx produced by
site-directed mutagenesis. Although
1 for authentic
porcine PHGPx is 0.07 µM s, and
2 is 8.3 µM s, the cysteine-containing PHGPx mutein, with a
1 of 20 and
2 of 40,000 µM s (13), resembles PfTPx.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Representative double-reciprocal Dalziel plot
for the PfTPx-catalyzed reduction of t-bOOH at five different
concentrations of GSH (3.3 mM ( ), 4.9 mM
( ), 6.6 mM ( ), 8.2 mM ( ), 10 mM ). Initial velocities and pertinent substrate
concentrations are obtained by the single curve progression analysis
according to Forstrom et al. (22). The ordinate
[E0] × t/([S0] [S])
represents [E0]/v, and the abscissa
is equivalent to 1/[ROOH]. Dalziel coefficients of the general
Dalziel equation for two-substrate reactions [E]/v = 0 + 1/[A] + 2/B + 1,2/[A][B] represent empirical parameters obtained
from slopes and intercepts of primary and secondary plots, as
indicated. Parallelity of the slopes indicate that the term
1,2/[A][B] is zero, as is typical for ping-pong
mechanisms (23).
|
|

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 4.
Secondary Dalziel plot for the
PfTPx-catalyzed reduction of t-bOOH by GSH. The reciprocal
apparent maximum velocities (n = 3 each) extrapolated
for infinite concentration of t-bOOH, are plotted against the
reciprocal GSH concentrations. The slope yields 2.
Cutting the ordinate at zero indicates that the term 0
approaches zero, which implies that the maximum velocity and
Km values of PfTPx are infinite for the pair of
substrates investigated.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Kinetic coefficients and apparent rate constants of PfTPx for the
reduction of different hydroperoxides by GSH
The Dalziel coefficients 1 and 2 of PfTPx are
calculated from three different sets of experiments. The extrapolated
coefficient 0 is zero within experimental error for all
reactions. 2 values for the GSH-dependent
reaction of PfTPx do not differ significantly, whereas 1 and
k1' values depend on the nature of the
hydroperoxide. CumeneOOH, cumene hydroperoxide; PCOOH,
phosphatidylcholine hydroperoxide; Sec, selenocysteine. Data for PHGPx
are taken from Maiorino et al. (13).
|
|
Thioredoxin as Reducing Substrate of PfTPx--
In view of the low
efficiency of PfTPx in GSH-dependent hydroperoxide
reduction and the sequence similarities to the less GSH-specific pGPx
(32) and PHGPx (33, 34), its activity with alternative reducing
substrates, notably thioredoxins, was investigated. Table
II shows that PfTPx indeed accepts
thioredoxins of various species. The turnover rates observed with
thioredoxin of E. coli and man in the submillimolar range
were similar to those measured with 10 mM GSH, and
autologous plasmodial thioredoxin at a concentration of only 5 µM triggered a significantly faster reaction.
View this table:
[in this window]
[in a new window]
|
Table II
Specificity of PfTPx for thioredoxins
GSH as reducing substrate was replaced by Trx of E. coli (85 µM), human (200 µM), and P. falciparum (5 µM), and glutathione reductase as
indicator enzyme (5.6 units/ml) was replaced by the autologous
thioredoxin reductases (0.5 unit/ml, 0.56 unit/ml, 0.001 unit/ml,
respectively). With each of the thioredoxins, the specific rate was at
least as high as that observed with GSH at 50-2000× the
concentration.
|
|
Based on these preliminary findings, the kinetics of PfTPx were
analyzed with thioredoxin (Trx) of P. falciparum (PfTrx) and t-bOOH. Fig. 5 reveals that also with
PfTrx a ping-pong pattern is observed. The secondary plot (Fig.
6), however, shows a marked difference
when compared with the kinetic pattern obtained with GSH as reducing
substrate. The slope no longer cuts the ordinate at zero, implying
Michaelis-Menten-type saturation kinetics with defined
2, Vmax, and
Km values. Also, the
2 value for PfTrx is much lower than for GSH (Table III). This kinetic pattern and
the coefficients obtained allow the following conclusions. (i) The
reducing substrate does not affect the reaction of the reduced enzyme
with the hydroperoxide (Equation 2);
1 or
k1, respectively, are not significantly
different for the GSH- and the PfTrx-driven reaction, as expected for a
ping-pong mechanism. (ii) Saturation kinetics therefore can only result
from a shift of rate constants in the reductive part of catalytic
cycle. Obviously, the formation of a complex between the oxidized
enzyme and PfTrx occurs faster with k+4
k
4 than its decay with
k+5 (Equation 6).
|
(Eq. 6)
|
(iii) The limiting Km value for the PfTrx is
defined by (k
4 + k+5)/k+4. It approximates
the dissociation constant Ks = k
4/k+4 and can be taken
as a measure of affinity for PfTrx. (iv) The overall rate determining
kcat, then, is k+5. (v)
The high k
value and saturation
kinetics with a low Km classify PfTrx as a specific
substrate of PfTPx.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Representative primary Dalziel plot of the
PfTPx-catalyzed reduction of t-bOOH by PfTrx. As in the
GSH-dependent reaction, parallel lines are observed with
1.5 ( ), 2.5 ( ), and 3.5 ( ) µM PfTrx, indicating
a ping-pong mechanism. The slopes ( ) do not differ significantly
from those obtained with GSH as reducing substrate.
|
|

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 6.
Secondary Dalziel plot of the PfTPx-catalyzed
reduction of t-bOOH by PfTrx. Data are based on duplicates of
primary plots, as exemplified in Fig. 7, plus two pilot measurements
performed at 5 µM PfTrx. The line cuts the ordinate at
0, which adopts a positive value with PfTrx as the
reducing substrate.
|
|
 |
DISCUSSION |
The glutathione peroxidase family of proteins is spread over the
whole living kingdom (2, 14). The name-giving classical glutathione
peroxidase, however, has so far only been detected in vertebrates,
where it proved to be a selenoprotein (35, 36) and of the related
selenoproteins, pGPx, gastrointestinal GPx, and PHGPx, only the latter
has been identified in a nonvertebrate species, Schistosoma
mansoni (37, 38). Genes encoding homologous proteins in which the
active site selenocysteine is replaced by cysteine appear to be widely
distributed in nature (2, 14). Such proteins are commonly addressed as
glutathione peroxidases, although their activity has never been
systematically analyzed. In fact, some representatives of the family
have been discovered in a biological context not reminiscent of an
antioxidant function, e.g. the cobalamine-binding protein in
E. coli (39), the salt stress-responsive protein in
Citrus plants (40), and androgen-responsive epidydymal proteins in mammals (41). Furthermore, any relevant peroxidase activity of such proteins may be questioned considering the
low efficiencies of cysteine-containing muteins of GPx and PHGPx.
To our knowledge, the efficiency and specificity of a naturally
occurring nonselenium GPx homologue is addressed here for the first
time. To this end, we have unfortunately to rely on an heterologously
expressed protein, since a purification of PfTPx from P. falciparum has not yet been feasible. In its basic
characteristics, however, the PfTPx gene expression product, as here
obtained, is presumed to closely resemble authentic PfTPx. Admittedly,
the discrepancy between the size of the deduced maximum sequence and the isolated protein is substantial. But this is also observed with the
closely related mammalian PHGPx. As in the PfTPx gene, two potential
start codons are contained in the PHGPx gene. In this case they are
alternatively used in a tissue-specific manner, either leading to
mitochondrial or cytosolic localization of the enzyme (42, 43). In each
case the processed expression product is the same (43), i.e.
a protein of about 19 kDa corresponding in size to a PfTPx starting
with Met-26. Monitoring the production of PfTPx in E. coli
does not reveal any primary product in the 24-kDa region. The
electrophoretic mobility of the band showing up upon induction is
identical to that of the isolated product (Fig. 1). Obviously, our
E. coli production strain has chosen the second ATG start
codon, which is also the preferred start codon of the mammalian PHGPx
genes. Certainly, the heterologously expressed PfTPx contains all
residues constituting the catalytic triad essential for hydroperoxide
reduction, i.e. Cys-76, Gln-111, and Trp-169 in homologous
position to Gln-81, selenocysteine 46, Trp-136 of porcine PHGPx (13,
24). Whether the missing N-terminal extension and the minor C-terminal
truncation affects specificity remains to be established. The gain of a
new specificity, as described here, is not likely explained by such modifications.
The low efficiency of PfTPx in reducing hydroperoxides does not
surprise but raises the question whether such low efficiency peroxidases may be implicated in antioxidant defense at all. The k
values for the reaction of PfTPx
with t-bOOH, cumene hydroperoxide, and H2O2
reported here come close to the k1' of the
cysteine mutein of porcine PHGPx with phosphatidylcholine hydroperoxide
(13). Similar k1' values were reported for
tryparedoxin peroxidase, a structurally unrelated peroxiredoxin also
working with sulfur catalysis (44). These observations suggest that,
with sulfur catalysis, rate constants near 104
M
1 s
1
can be reached for the reduction of hydroperoxides by thiols, whereas
107 M
1
s
1 are commonly observed with selenium
catalysis. Rate constants beyond 106
M
1 s
1
are also reported for heme-catalyzed hydroperoxide reduction (45). Less
efficient hydroperoxidases can reasonably be implicated in antioxidant
defense if their low molar efficiency is compensated for by extreme
concentration, as has been proposed for the hydroperoxide detoxification by a peroxiredoxin in trypanosomes (44). In line with
these considerations, E. coli overexpressing PfTPx proved to
be slightly more resistant to oxidative
challenge,2 which is not
surprising in view of PfTPx being the prominent protein in such cells
(Fig. 1). At less abundant levels, the ability of PfTPx to balance
oxidative stress may be doubted.
The observation that a GPx homologue reacts with thiols other than GSH
is not surprising either. Only for the cytosolic GPx has a pronounced
specificity for GSH been documented (46). This specificity is
considered to be due to basic residues, Arg-57, Arg-102, Arg-184,
Arg-185, and Lys-92 in bovine cGPx, which direct the SH group of the
substrate to the active-site selenium atom by electrostatic forces
(47). These residues are only partially conserved in the
gastrointestinal and extracellular isozymes and completely lost in
PHGPx-type enzymes. Accordingly, pGPx has been reported to accept
thioredoxin and glutaredoxin (32), and PHGPx, in the absence of GSH,
can form high molecular weight protein aggregates that are cross-linked
by Se-S and/or S-S bridges, a process shown to be of physiological
importance in late phases of mammalian sperm maturation (33). It does,
however, not react with E. coli thioredoxin and human
thioredoxin 1 and 4.3 In
contrast, PfTPx appears to be specialized for interaction with
thioredoxin, as evident from the kinetic data reported.
Nevertheless, a competition of GSH with thioredoxin for oxidized PfTPx
under in vivo conditions cannot be fully ruled out. Irrespective of uncertainties about the concentrations of GSH and Trx
in the various differentiation states of the parasites, the kinetic
parameters of PfTPx imply that the GSH-driven reaction falls short
under most conditions that could be envisaged to be physiologically
relevant, as is easily calculated by means of the rate equation
(Equation 1) and the kinetic coefficients (Table III). The PfTrx-driven
H2O2 reduction reaches apparent maximum velocities in the µM range of PfTrx. With 10 mM GSH, which may be taken as an upper physiological level,
the maximum velocities achieved with PfTrx are never reached. In fact,
the GSH-dependent reaction would break even with that of
PfTrx if the GSH levels were raised beyond 26 mM. At high
peroxide challenge, however, cellular GSH concentrations of 5-10
mM, which can not be rated as uncommon, may compete with
PfTrx if present in concentrations below the Km of
10 µM. These consideration are, however, not meant to
implicate a pivotal role of PfTPx in GSH-dependent hydroperoxide detoxification in P. falciparum. Irrespective
of the donor substrate, the efficiency of PfTPx in hydroperoxide reduction is limited by the low k1' values. The
GSH-dependent hydroperoxide removal by cGPx in the
parasite's host cells is faster by orders of magnitude (48). The
specificity of PfTPx for thioredoxin points to biological roles
distinct from antioxidant defense such as redox regulation of gene
expression and differentiation processes, as are attributed to
PHGPx and low efficiency peroxiredoxin-type peroxidases in mammalian
cells (33, 49-51).
View this table:
[in this window]
[in a new window]
|
Table III
Compilation of kinetic data of PfTPx with GSH and PfTrx as reducing
substrate
For the GSH-driven reaction, 2 values were averaged from all
experiments irrespective of the peroxide substrate. 1 value
and k1' values are those for t-bOOH.
|
|
The search for other candidates that exert antioxidant defense in
Plasmodia species therefore remains rewarding. An
Fe-superoxide dismutase and catalase are reportedly present in
Plasmodia species. The increase of alkyl hydroperoxide
reduction by supplementation of cultures of malaria parasites with
selenium (52), however, needs clarification. It cannot be ruled out
that selenium, when provided as selenite or selenocystine in micromolar
concentrations, is unspecifically incorporated into PfTPx to some
extent, and selenium incorporation into 1% PfTPx could result in the
observed duplication of turnover, taking into account the pronounced
difference of rate constants between Se- and non-Se glutathione
peroxidases (12, 13). Alternatively, also in Plasmodia
species real selenoperoxidases might exist that could be typical
glutathione peroxidases or peroxiredoxins. Peroxiredoxin-type putative
thioredoxin peroxidases have recently been identified in
Plasmodia (GenBankTM accession number AF225977
and AF225978) and are currently being characterized. In mammals, a
member of this family has been reported to be a non-Se GPx (53), and in
amino acidophilic bacteria, such enzymes are selenoproteins (54). Thus,
many options to link selenium-enhanced
glutathione-dependent peroxide metabolism to antioxidant
defense in Plasmodium remain to be explored. PfTPx, being a
low efficiency nonselenium peroxidase with a clear preference for the
pleiotropic redox regulator thioredoxin, is not the ideal candidate to
play this role.
 |
ACKNOWLEDGEMENTS |
We thank Claudia Wylegalla for
technical help in enzyme preparation and activity measurements and Dr.
Josef Wissing for performing the MALDI-TOF analysis.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§§
To whom correspondence should be addressed. Tel.:
49-531-6181599; Fax: 49-531-6181458; E-mail: lfl@gbf.de.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008631200
2
B. Gamain, unpublished observations.
3
Brigelius-Flohé and L. Flohé,
unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
GPx, glutathione
peroxidase;
pGPx, plasma GPx;
cGPx, cytosolic GPx;
PHGPx, phospholipidhydroperoxide GPx;
PfTPx, thioredoxin peroxidase of
P. falciparum;
Trx, thioredoxin;
PfTrx, Trx of P. falciparum;
MALDI-TOF, matrix-assisted laser desorption and
ionization time of flight mass spectrometry;
t-bOOH, t-butyl
hydroperoxide.
 |
REFERENCES |
1.
|
Fairfield, A. S.,
Abosch, A.,
Ranz, A.,
and Eaton, J. W.
(1988)
Mol. Biochem. Parasitol.
30,
77-82[Medline]
[Order article via Infotrieve]
|
2.
|
Gamain, B.,
Langsley, G.,
Fourmaux, M. N.,
Touzel, J. P.,
Camus, D.,
Dive, D.,
and Slomianny, C.
(1996)
Mol. Biochem. Parasitol.
78,
237-248[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Ayi, K.,
Cappadoro, M.,
Branca, M.,
Turrini, F.,
and Arese, P.
(1998)
FEBS Lett.
424,
257-261[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Luersen, K.,
Walter, R. D.,
and Müller, S.
(1999)
Mol. Biochem. Parasitol.
98,
131-142[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Farber, P. M.,
Becker, K.,
Müller, S.,
Schirmer, R. H.,
and Franklin, R. M.
(1996)
Eur. J. Biochem.
239,
655-661[Abstract]
|
6.
|
Malhotra, K.,
Salmon, D.,
Le Bras, J.,
and Vilde, J. L.
(1988)
Infect. Immun.
56,
3305-3309[Medline]
[Order article via Infotrieve]
|
7.
|
Jensen, J. B.,
and Vande Waa, J. A.
(1988)
J. Immunol. Methods
112,
201-205[Medline]
[Order article via Infotrieve]
|
8.
|
Atamna, H.,
and Ginsburg, H.
(1993)
Mol. Biochem. Parasitol.
61,
231-241[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Buffinton, G. D.,
Hunt, N. H.,
Cowden, W. B.,
and Clark, I. A.
(1988)
Biochem. J.
249,
63-68[Medline]
[Order article via Infotrieve]
|
10.
|
Schwarzer, E.,
Müller, O.,
Arese, P.,
Siems, W. G.,
and Grune, T.
(1996)
FEBS Lett.
388,
119-122[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Flohé, L.,
Hecht, H. J.,
and Steinert, P.
(1999)
Free Radic. Biol. Med.
27,
966-984[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Rocher, C.,
Lalanne, J.,
and Chaudiere, J.
(1992)
Eur. J. Biochem.
205,
955-960[Abstract]
|
13.
|
Maiorino, M.,
Aumann, K-D.,
Brigelius-Flohé, R.,
Doria, D.,
van den Heuvel, J.,
McCarthy, J.,
Roveri, A.,
Ursini, F.,
and Flohé, L.
(1995)
Biol. Chem. Hoppe-Seyler
376,
651-660[Medline]
[Order article via Infotrieve]
|
14.
|
Ursini, F.,
Maiorino, M.,
Brigelius-Flohé, R.,
Aumann, K. D.,
Roveri, A.,
Schomburg, D.,
and Flohé, L.
(1995)
Methods Enzymol.
252,
38-53[Medline]
[Order article via Infotrieve]
|
15.
|
Butcher, L. A.,
and Tomkins, J. K.
(1985)
Anal. Biochem.
148,
348-388
|
16.
|
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Flohé, L.
(1988)
in
CRC Handbook of Free Radicals and Antioxidants in Biomedicine
(Miquel, J.
, Quintanilha, A. T.
, and Weber, H., eds), Vol. III
, pp. 281-286, CRC Press, Inc., Boca Raton, FL
|
18.
|
Maiorino, M.,
Gregolin, C.,
and Ursini, F.
(1990)
Methods Enzymol.
186,
448-457[Medline]
[Order article via Infotrieve]
|
19.
|
Maiorino, M.,
Wissing, J. B.,
Brigelius-Flohé, R.,
Calabrese, F.,
Roveri, A.,
Steinert, P.,
Ursini, F.,
and Flohé, L.
(1998)
FASEB J.
12,
1359-1370[Abstract/Free Full Text]
|
20.
|
Kanzok, S. M.,
Schirmer, R. H.,
Turbachova, I.,
Iozef, R.,
and Becker, K.
(2000)
J. Biol. Chem.
275,
40180-40186[Abstract/Free Full Text]
|
21.
|
Müller, S.,
Gilberger, T. W.,
Farber, P. M.,
Becker, K.,
Schirmer, R. H.,
and Walter, R. D.
(1996)
Mol. Biochem. Parasitol.
80,
215-219[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Forstrom, J. W.,
Stults, F. H.,
and Tappel, A. L.
(1979)
Arch. Biochem. Biophys.
193,
51-55[Medline]
[Order article via Infotrieve]
|
23.
|
Dalziel, K.
(1957)
Acta Chem. Scand.
11,
1706-1723
|
24.
|
Brigelius-Flohé, R.,
Aumann, K.-D.,
Blöcker, H.,
Gross, G.,
Kiess, M.,
Kloeppel, K.- D.,
Maiorino, M.,
Roveri, A.,
Schuckelt, R.,
Ursini, F.,
Wingender, E.,
and Flohé, L.
(1994)
J. Biol. Chem.
10,
7342-7348
|
25.
|
Epp, O.,
Ladenstein, R.,
and Wendel, A.
(1983)
Eur. J. Biochem.
133,
51-69[Medline]
[Order article via Infotrieve]
|
26.
|
Ren, B.,
Huang, W.,
Akesson, B.,
and Ladenstein, R.
(1997)
J. Mol. Biol.
268,
869-885[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Flohé, L.,
Aumann, K-D.,
Brigelius-Flohé, R.,
Schomburg, D.,
Strassburger, W.,
and Ursini, F.
(1993)
in
Active Oxygens, Lipid Peroxides, and Antioxidants
(Yagi, K., ed)
, pp. 291-311, CRC Press, Inc., Boca Raton, FL
|
28.
|
Flohé, L.,
Loschen, G.,
Günzler, W. A.,
and Eichele, E.
(1972)
Hoppe-Seyler's Z. Physiol. Chem.
353,
987-999[Medline]
[Order article via Infotrieve]
|
29.
|
Günzler, W. A.,
Vergin, H.,
Müller, I.,
and Flohé, L.
(1972)
Hoppe-Seyler's Z. Physiol. Chem.
353,
1001-1004[Medline]
[Order article via Infotrieve]
|
30.
|
Ursini, F.,
Maiorino, M.,
and Gregolin, C.
(1985)
Biochim. Biophys. Acta
839,
62-70[Medline]
[Order article via Infotrieve]
|
31.
|
Esworthy, R. S.,
Chu, F. F.,
Geiger, P.,
Giroti, A. W.,
and Doroshow, J. H.
(1993)
Arch. Biochem. Biophys.
307,
29-34[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Björnstedt, M.,
Xue, J.,
Huang, W.,
Akesson, B.,
and Holmgren, A.
(1994)
J. Biol. Chem.
269,
29382-29384[Abstract/Free Full Text]
|
33.
|
Ursini, F.,
Heim, S.,
Kiess, M.,
Maiorino, M.,
Roveri, A.,
Wissing, J.,
and Flohé, L.
(1999)
Science
5432,
1393-1396[CrossRef]
|
34.
|
Godeas, C.,
Tramer, F.,
Micali, F.,
Roveri, A.,
Maiorino, M.,
Nissi, C.,
Sandri, G.,
and Panfili, E.
(1996)
Biochem. Mol. Med.
59,
118-124[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Rotruck, J. T.,
Pope, A. L.,
Ganther, H. E.,
Swanson, A. B.,
Hafeman, D. G.,
and Hoekstra, W. G.
(1973)
Science
179,
588-590[Medline]
[Order article via Infotrieve]
|
36.
|
Flohé, L.,
Günzler, W. A.,
and Schock, H. H.
(1973)
FEBS Lett.
32,
132-134[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Williams, D. L.,
Pierce, R. J.,
Cookson, E.,
and Capron, A.
(1992)
Mol. Biochem. Parasitol.
52,
127-130[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Maiorino, M.,
Roche, C.,
Kiess, M.,
Koenig, K.,
Gawlik, D.,
Matthes, M.,
Naldini, E.,
Pierce, R.,
and Flohé, L.
(1996)
Eur. J. Biochem.
238,
838-844[Abstract]
|
39.
|
Friedrich, M. J.,
DeVeaux, L. C.,
and Kachner, R. J.
(1986)
J. Bacteriol.
167,
928-934[Medline]
[Order article via Infotrieve]
|
40.
|
Holland, D.,
Ben-Hayyim,
Falin, Z.,
Camoin, L.,
Strosberg, A. D.,
and Eshdat, Y.
(1993)
Plant Mol. Biol.
21,
923-927[Medline]
[Order article via Infotrieve]
|
41.
|
Ghyselinck, N. B.,
and Dufaure, J-P.
(1990)
Nucleic Acids Res.
18,
7144[Medline]
[Order article via Infotrieve]
|
42.
|
Pushpa-Rekha, T.,
Burdsal, L. M.,
Oleksa, L. M.,
Chisolm, G. M.,
and Driscoll, D. M.
(1995)
J. Biol. Chem.
270,
26993-26999[Abstract/Free Full Text]
|
43.
|
Arai, M.,
Imai, H.,
Sumi, D.,
Imanaka, T.,
Takano, T.,
Chiba, N.,
and Nakagawa, Y.
(1996)
Biochem. Biophys. Res. Commun.
227,
433-439[CrossRef][Medline]
[Order article via Infotrieve]
|
44.
|
Nogoceke, E.,
Gommel, D. U.,
Kiess, M.,
Kalisz, H. M.,
and Flohé, L.
(1997)
Biol. Chem.
378,
827-836[Medline]
[Order article via Infotrieve]
|
45.
|
Chance, B.,
Sies, H.,
and Boveris, A.
(1979)
Physiol. Rev.
59,
527-605[Free Full Text]
|
46.
|
Flohé, L.,
Günzler, W. A.,
Jung, G.,
Schaich, E.,
and Schneider, F.
(1971)
Hoppe-Seyler's Z. Physiol. Chem.
352,
159-169[Medline]
[Order article via Infotrieve]
|
47.
|
Aumann, K.-D.,
Bedorf, N.,
Brigelius-Flohé, R.,
Schomburg, D.,
and Flohé, L.
(1997)
Biomed. Environ. Sci.
10,
136-155[Medline]
[Order article via Infotrieve]
|
48.
|
Flohé, L.
(1979)
CIBA Found. Symp.
65,
95-122
|
49.
|
Brigelius-Flohé, R.,
Friedrichs, B.,
Maurer, S.,
Schultz, M.,
and Streicher, R.
(1997)
Biochem. J.
328,
199-203[Medline]
[Order article via Infotrieve]
|
50.
|
Jin, D.-Y.,
Chae, H. Z.,
Rhee, S. G.,
and Jeang, K.-T.
(1997)
J. Biol. Chem.
272,
30952-30961[Abstract/Free Full Text]
|
51.
|
Flohé, L.,
Andreesen, J. R.,
Brigelius-Flohé, R.,
Maiorino, M.,
and Ursini, F.
(2000)
IUBMB Life
49,
411-420[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Gamain, B.,
Arnaud, J.,
Favier, A.,
Camus, D.,
Dive, D.,
and Slomianny, C.
(1996)
Free Radic. Biol. Med.
21,
559-565[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Chen, J.-W.,
Dodia, C.,
Feinstein, S. I.,
Jain, M. K.,
and Fisher, A. B.
(2000)
J. Biol. Chem.
275,
28421-28427[Abstract/Free Full Text]
|
54.
|
Andreesen, J. R.,
Wagner, M.,
Sonntag, D.,
Kohlstock, M.,
Harms, C.,
Gursinsky, T.,
Jäger, J.,
Parther, T.,
Kabisch, U.,
Gräntzdörffer, A.,
Pich, A.,
and Söhling, B.
(1999)
Biofactors
10,
263-270[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.