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7/31/2019 O parasita da malria Plasmodium falciparum possui um funcional
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Molecular & Biochemical Parasitology 112 (2001) 219228
The malaria parasite Plasmodium falciparum possesses a functionalthioredoxin system
Zita Krnajski, Tim-W. Gilberger 1, Rolf D. Walter, Sylke Muller *
Bernhard Nocht Institute for Tropical Medicine, Biochemical Parasitology, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany
Received 10 August 2000; received in revised form 2 November 2000; accepted 13 November 2000
Abstract
The thioredoxin system consists of the NADPH dependent disulphide oxidoreductase thioredoxin reductase (TrxR) which
catalyses the reduction of the small protein thioredoxin. This system is involved in a variety of biological reactions including the
reduction of deoxyribonucleotides, transcription factors and hydrogen peroxide. In recent years the TrxR of the malaria parasite
Plasmodium falciparum was isolated and characterised using model substrates like 5,5%-dithiobis (2-nitrobenzoic acid) (DTNB) and
Escherichia coli thioredoxin. Here we report on the isolation of a cDNA encoding for P. falciparum thioredoxin (PfTrx) and the
expression and characterisation of the recombinant protein, the natural substrate of PfTrxR. The deduced amino acid sequence
of PfTrx encodes for a polypeptide of 11 715 Da and possesses the typical thioredoxin active site motif CysGlyProCys. Both
cysteine residues are essential for catalytic activity of the protein, as shown by mutational analyses. Steady state kinetic analyses
with PfTrxR and PfTrx in several coupled assay systems resulted in Km-values for PfTrx in the range of 0.82.1 mM which is
about 250-fold lower than for the model substrate E. coli thioredoxin. Since the turnover of both substrates is similar, the catalytic
efficiency of PfTrxR to reduce the isolated PfTrx is at least 250-fold higher than to reduce E. coli thioredoxin. PfTrx containsa cysteine residue in position 43 in addition to the active-site cysteine residues, which is partially responsible for dimer formation
of the protein as demonstrated by changing this amino acid into an alanine residue. Using DTNB we showed that all three
cysteine residues present in PfTrx are accessible to modification by this compound. Surprisingly the first cysteine residue of the
active site motif (Cys30) is less accessible than the second cysteine (Cys33), which is highly prone to the modification. These results
suggest a difference in the structure and reaction mechanism of PfTrx compared to other known thioredoxins. 2001 Elsevier
Science B.V. All rights reserved.
Keywords: Plasmodium falciparum ; Thioredoxin reductase; Redox system; Malaria; Oxidative stress
www.parasitology-online.com
1. Introduction
The thioredoxin system consists of the NADPH de-
pendent disulphide oxidoreductase thioredoxin reduc-
tase (TrxR) and the small protein thioredoxin. This
system is responsible for several redox reactions withinthe cell and thioredoxins are regarded as general redox
messengers that interact with a wide variety of proteins.
Thioredoxins possess a typical CysGlyProCys active
site motif. In the oxidised state the cysteine residues
form a disulphide which is reduced by thioredoxin
reductase. In the reduced state one of the active site
cysteines of thioredoxin interacts with enzymes such as
ribonucleotide reductase, 3%-phospho-adenylylsulfate re-
ductase and methionine sulfoxide reductase [1 4]. In
addition it has been shown that thioredoxins are in-
volved in transcriptional control by modifying the re-dox state of thiols in the active site of transcription
factors and altering their activation state. Transcription
factors regulated by thioredoxins are OxyR, NF-kB
Abbre6iations: DTNB, 5,5%-dithiobis (2-nitrobenzoic acid); DTT,
dithiothreitol; PfTrx, P. falciparum thioredoxin; PfTrxR, P. falci-
parum thioredoxin reductase; TNB; 5%-thionitrobenzoic acid.
* Corresponding author. Present address: Department of Biochem-
istry, Wellcome Trust Biocentre, University of Dundee, Dundee DD1
5EH, Scotland, UK. Tel.: +49-40-42818344; fax: +49-40-42818418.
E-mail address: sylkemueller@yahoo.com (S. Muller). Note: Nucleotide sequence data reported in this paper are avail-
able in the EMBL, GenBankTM and DDJB databases under the
accession number CAB90828.1 Present address: The Walter and Eliza Hall Institute of Medical
Research, PO Royal Melbourne Hospital, Melbourne 3050, Aus-
tralia.
0166-6851/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 6 8 5 1 ( 0 0 ) 0 0 3 7 2 - 8
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Z. Krnajski et al. /Molecular & Biochemical Parasitology 112 (2001) 219228220
and AP-1 [5 9]. A third function of the thioredoxin
system which has only been discovered in recent years
is the reduction of reactive oxygen species which is
performed by an interaction of thioredoxins and perox-
iredoxins. This increasing family of proteins has been
identified in a wide variety of organisms and its abun-
dance in the cell has led to the suggestion that it
represents one of the major peroxide detoxifyingproteins [1012]. It certainly plays an important role in
helminths where it was suggested to be the key enzy-
matic system to deal with hydrogen peroxide [13]. In
the human malaria parasite Plasmodium falciparum,
glutathione peroxidase is present but appears to have a
very low efficiency for the reduction of hydrogen perox-
ide [1416] and the thioredoxin system is proposed to
be the main detoxification system for reactive oxygen
species. It is well established that Plasmodium infected
erythrocytes are under enhanced oxidative stress. How-
ever, the parasite-host cell unit appears to be able to
maintain the necessary balance between oxidants and
antioxidants so that parasite development is not im-
paired. Several enzymatic antioxidants have been iso-
lated from the parasites [14,17,18] but their role for
parasite survival awaits evaluation. We have isolated
and partially characterised TrxR from P. falciparum
[1922] and have now identified a thioredoxin-like se-
quence in the P. falciparum genome database (TIGR).
Here we report on the isolation, recombinat expression
and characterisation of this thioredoxin-like protein
which is highly active with P. falciparum thioredoxin
reductase (PfTrxR) and may represent the link to thereduction of other essential cellular components in the
parasite such as peroxiredoxins to confer reduction of
hydrogen peroxide.
2. Materials and methods
2.1. Material
Escherichia coli thioredoxin was a kind gift from
Professor Charles H. Williams Jr., Ann Arbor, USA,
and the expression vector pJC40 was a gift from Dr
Joachim Clos, Hamburg, Germany. The pcDNAII li-
brary of P. falciparum was a kind gift from Professor
David Kaslow, Bethesda, USA. Bovine insulin and
5,5%-dithiobis (2-nitrobenzoic acid) (DTNB) were pur-
chased from Sigma. NADPH was from Boehringer
Mannheim. Recombinant TrxR from P. falciparum was
prepared as described in Gilberger et al. [21].
2.2. Isolation of a P. falciparum thioredoxin-like
sequence and expression of the recombinant protein
Preliminary sequence data for P. falciparum chromo-
some 14 was obtained from The Institute for Genomic
Research website (www.tigr.org). Sequencing of chro-
mosome 14 was part of the International Malaria
Genome Sequencing Project and was supported by
awards from the Burroughs Wellcome Fund and the
U.S. Department of Defense.
Using the sequence specific sense oligonucleotide 5%-
GCGCGCATATGGTAAAAATTGTAACTAGTC-3%
coding for the first seven amino acids of the potentialP. falciparum thioredoxin and the antisense oligonucle-
otide 5%-GCGCGCTCGAGTTAAGCTGCGTATTT-
TTCG-3% encoding the last six amino acids of the
potential coding region of the genomic sequence, a 315
bp fragment was amplified from a cDNA plasmid
library as a template (pcDNA II). The sense primer
contained an NdeI restriction site and the antisense
primer contained an XhoI restriction site to facilitate
directional cloning into the expression plasmid pJC40
previously digested with NdeI and XhoI. The thiore-
doxin coding region was amplified using Pfu poly-
merase (Stratagene) under the following conditions:
95C for 2 min, 95C for 30 s, 50C for 30 s and 68C
for 1 min. The PCR fragment was gel purified, digested
with NdeI and XhoI, subcloned into pJC40 and the
sequence was determined using the Sanger dideoxy
termination method [23].
The expression plasmid containing the open reading
frame of PfTrx was transformed into E. coli BL 21
(DE3) (Stratagene). A single colony was picked and an
overnight culture in Luria-Bertani medium containing
50 mg ml1 ampicillin was inoculated. The bacterial
culture was diluted 1:100 into Terrific Broth containing50 mg ml1 ampicillin and grown in a 2 l fermenter
(Braun-Melsungen) at 37C until the OD600 reached 2.0
before expression of PfTrx was induced by addition of
1.0 mM isopropyl-b-D-thiogalactopyranoside. The tem-
perature was reduced to 25C after induction to prevent
the formation of inclusion bodies during expression of
the recombinant protein. After the cells reached an
OD600 of about 20 they were harvested and resuspended
in 50 mM TrisHCl buffer pH 7.9 containing 100 mM
NaCl, 40 mM imidazol and 1 mM dithiothreitol (DTT)
and stored at 20C.
The protein was purified using Ni2+-chelating chro-
matography according to the manufacturers recom-
mendation (Qiagen). Protein concentration was
calculated by using the molar extinction coefficient of
13 700 M1 cm1 for E. coli thioredoxin at 280 nm.
The purity of the protein was assessed by SDS-PAGE.
2.3. Site directed mutagenesis
To identify the redox active residues of PfTrx, Cys30
and 33 were replaced by alanine residues according to
[20,21]. The mutagenic oligonucleotides used were sense5%-GCTGAATGGGCTGGACCATGCAAAAG-3% and
antisense 5%-CTTTTGCATGGTCCAGCCCATTCA-
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Z. Krnajski et al. /Molecular & Biochemical Parasitology 112 (2001) 219228 221
GC-3% to replace Cys30 by alanine and sense
5%-GAATGGTGTGGACCAGCCAAAAGAATTGC-
CCC-3% and antisense 5%-GGGGCAATTCTTTTG-
GCTGGTCCACACCATTC-3% to exchange Cys33 by
alanine (nucleotides underlined show mutated/altered
positions). Further, the third cysteine residue (Cys43) of
P. falciparum thioredoxin was replaced by alanine to
determine whether it is responsible for dimer formationof the wild-type protein. The mutagenic oligonucle-
otides were: sense 5%-CCCATTTTATGAAGAAGC-
CTCCAAAACATACAC-3% and antisense 5%-GTG-
TATGTTTTGGAGGCTTCTTCATAAAATGGG-3%.
All mutations were verified by nucleotide sequencing
[24]. The plasmids (pJC40) containing the mutated
open reading frames were transformed into E. coli BL
21 (DE3) and expression and purification was per-
formed as described above for the wild-type protein.
2.4. Enzyme assays
To establish that the recombinant PfTrxWT and
PfTrxC43A mutant are substrates of PfTrxR several
distinct enzyme assays using different acceptor
molecules for thioredoxin were performed and the ki-
netic parameters were compared. The insulin assay
mixture contained 100 mM Hepes pH 7.6, 0.2 mM
EDTA, 200 mM NADPH, 110 mM PfTrx orPfTrxC43A and 2 mg ml1 insulin and the change in
absorbance at 340 nm was determined. The DTNB
assay mixture contained essentially the same compo-
nents as described above but instead of insulin, 40 mMDTNB was added and absorbance at 412 nm was
followed. In order to test a natural substrate for the
reduction by thioredoxin we used a recombinantly ex-
pressed potential thioredoxin peroxidase (peroxire-
doxin) in a third assay system. The assay mixture
contained 100 mM Hepes pH 7.6, 0.2 mM EDTA, 200
mM NADPH, 110 mM thioredoxin and 500 mg ml1
P. falciparum peroxiredoxin (Muller et al., unpublished
data).
The Km-values, turnover numbers and catalytic effi-
ciencies for PfTrxR and the respective substrates under
the different assay conditions were determined in dupli-
cate using 35 independent protein preparations. Stan-
dard deviations were calculated using the computer
software Excel. In comparision E. coli Trx was used as
a substrate for PfTrxR in the insulin assay.
In order to establish that the two potential redox
active cysteine residues (Cys30 and 33) interact with
either PfTrxR and the acceptor molecule we used both
mutant proteins PfTrxC30A and PfTrxC33A in our
activity assays described above.
2.5. Thiol accessibility
To investigate whether all three cysteine residues
present in PfTrx (Cys30, 33 and 43) are accessible to
solvent, PfTrxWT and mutant proteins were modified
with DTNB. Fifty micromolar of either PfTrxWT,
PfTrxC30A, PfTrxC33A or PfTrxC43A were treated
with a 10-fold molar excess of DTT to fully reduce the
sulfhydryl groups of the proteins. Subsequently, the
samples were dialysed overnight against 2 l of 50 mM
potassium phosphate buffer pH 7.6 containing 1 mMEDTA and then reacted with a 5-fold molar excess of
DTNB to modify the free thiol-groups of the proteins.
During this reaction 5%-thionitrobenzoic acid (TNB
anions) should be released when free thiol groups are
oxidised. The number of accessible thiols can be calcu-
lated by using the molar extinction coefficient of 13 600
M1 cm1 at 412 nm for TNB. After modification
the proteins were separated from non-reacted DTNB
and free TNB by gel filtration on a Sephadex G-25
column (Pharmacia), previously equilibrated with 50
mM potassium phosphate buffer pH 7.6 containing 1
mM EDTA. The fractions containing TNB-modified
proteins and free TNB were identified by absorption
spectrophotometry (240580 nm).
3. Results
3.1. Sequence analysis
Using PCR the coding region of the putative PfTrx
was amplified. The deduced amino acid sequence en-codes for a polypeptide of 104 amino acids and a
calculated molecular mass of 11 715 Da. The sequence
has the highest degree of identity with the thioredoxins
II of Schizosaccharomyces pombe and Saccharomyces
cere6isiae (51 and 49%, respectively), whereas it has
only a moderate degree of identity with the E. coli
thioredoxin I amino acid sequence (34%). PfTrx con-
tains the typical thioredoxin motif CysGlyProCys (Fig.
1), representing the redox active cysteine residues.
Apart from this active site motif, other residues are
conserved in almost all known thioredoxin sequences,
like Pro73, which is equivalent to Pro76 in the E. coli
thioredoxin. This residue is involved in the formation of
cis peptide bonds which stabilize the bacterial protein
and may have the same function in the parasite thiore-
doxin [2426]. In addition a third cysteine residue was
identified in position 43 which may be involved in the
formation of protein dimers in vitro and in vivo (Fig.
2).
3.2. Expression and purification of recombinant P. falci-
parum thioredoxin
PfTrx wild-type and mutant proteins were expressed
in E. coli BL 21 (DE3) as His-tagged fusion proteins
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Z. Krnajski et al. /Molecular & Biochemical Parasitology 112 (2001) 219228222
Fig. 1. Alignment of P. falciparum thioredoxin amino acid sequence with thioredoxins of other organisms. Trx P.f.: thioredoxin of P. falciparum;
Trx II S. p.: thioredoxin II of S. pombe; Trx II S. c.: thioredoxin II of S. cere6isiae; Trx I S. c.: thioredoxin I of S. cere6isiae; Trx H. s.:
thioredoxin of Homo sapiens; Trx I E. c.: thioredoxin I of E. coli. (*): amino acids identical to P. falciparum thioredoxin. (): gaps introduced
to obtain the best alignment.
which allows purification by Ni2+-chelating
chromatography. During the purification procedure 1
mM DTT was added to all buffers, except the elution
buffer, to avoid precipitation of the proteins. Even
under these conditions the yield from the purificationwas largely diminished by constant precipitation of the
proteins. According to gel filtration on Sephadex S-75
PfTrx is active as a monomer of about 11 kDa which is
in good agreement with the predicted molecular mass
of the deduced amino acid sequence and the molecular
mass determined by reducing SDS-PAGE (Fig. 3).
However, analysis by SDS-PAGE under non-reducing
conditions revealed that the addition of
b-mercaptoethanol is required to fully reduce possible
dimers formed during the purification procedure and
that these dimers are partly attributable to theformation of intermolecular disulphide bridges (Fig. 2).
3.3. Modification of P. falciparum thioredoxin wild-type
and mutant proteins with DTNB
PfTrxWT, PfTrxC30A, PfTrxC33A and PfTrxC43A
were modified with DTNB which resulted in the forma-
tion of PfTrxWT-TNB, PfTrxC33-TNB and
PfTrxC30-TNB mixed disulphides, respectively. The
concentration of TNB released during this reaction
was calculated by determining the absorbance at 412
nm using the extinction coefficient of 13 600 M1
cm1 for TNB [27].PfTrxWT, PfTrxC30A, PfTrxC33A and PfTrxC43A
exhibit symmetrical absorption peaks at 280 nm
whereas the modified proteins PfTrxWT, PfTrxC30-
TNB and PfTrxC33-TNB develop pronounced shoul-
ders around 324 nm, respectively (Fig. 4 A D).PfTrxRC43A shows no modification after treatment
with DTNB because there are no free thiols accessible
in this protein species (Fig. 4 D). To obtain full modifi-
cation the proteins were incubated with DTNB
overnight and then the modified protein species wereseparated from excess DTNB and formed TNB by gel
filtration. All fractions were analysed by absorption
spectroscopy (240 580 nm) and the concentration of
released TNB was calculated (Table 1). DTNB treat-
ment with PfTrxWT results in the release of 0.71 M
equivalents of TNB, suggesting that there is one ac-
cessible thiol in this protein species. According to the
deduced amino acid sequence we assume that this reac-
tion is attributable to modification of Cys43, since
Cys30 and 33 should form a disulphide in the wild-type
protein. Treating PfTrxC43A with DTNB only led to a
slight production of TNB, which is most likely due to
the spontaneous reduction of DTNB during the incuba-
Fig. 2. SDS-PAGE of P. falciparum thioredoxin wild-type and P.
falciparum thioredoxin C43A. Lane 1: 5 mg of PfTrxWT without
addition of b-mercaptoethanol. Lane 2: 3 mg of PfTrxC43A without
addition of b-mercaptoethanol. Lane 3: PfTrxWT with addition of
2.5% (v/v) b-mercaptoethanol. Lane 4: PfTrxC43A with addition of
2.5% (v/v) b-mercaptoethanol. Molecular mass standards: 10 kDaladder. Proteins were resolved on a 15% SDS-PAGE and visualized
with coomassie brilliant blue.
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Z. Krnajski et al. /Molecular & Biochemical Parasitology 112 (2001) 219228 223
Fig. 3. SDS-PAGE of P. falciparum thioredoxin wild-type and P.
falciparum thioredoxin C30A and P. falciparum thioredoxin C33A.
M: Molecular mass standards, 10 kDa ladder. Lane 1: 1 00 000g
supernatant of E. coli BL21 (DE3) containing the expression plasmid
of PfTrxWT (3 mg). Lane 2: 1 mg of PfTrxWT after purification with
Ni2+-chelating chromatography. Lane 3: 1 mg of PfTrxC30A after
purification with Ni2+-chelating chromatography. Lane 4: 1 mg of
PfTrxC33A after purification with Ni2+-chelating chromatography.
Proteins were resolved on a 15% SDS-PAGE and visualized with
coomassie brilliant blue.
units mg1 depending on the assay performed. These
differences are possibly due to the different ability ofPfTrx to interact with different acceptor molecules used
in the assays. Obviously DTNB represents a poor ac-
ceptor for the reducing equivalents of PfTrx orPfTrxC43A, although it has been reported to interact
perfectly well with E. coli thioredoxin [28]. In compari-
son E. coli thioredoxin was used as a substrate forPftrxR in the insulin assay and the Km-value deter-
mined was 500 mM with a kcat of 1688 min1.
Both mutant proteins PfTrxC30A and PfTrxC33A
were incapable of reacting with PfTrxR in any of the
enzyme assays performed. We also used both,PfTrxC30A and PfTrxC33A at 5 mM, as potential
inhibitors of the PfTrxRPfTrx reaction, but the re-
duction was not impaired by the mutant proteins (Fig.
5).
4. Discussion
The thioredoxin redox system has attracted a lot of
interest in the recent years. One reason is the fact that
this system is responsible for a wide variety of redox
reactions within the cell and is involved in redox con-
trol and signalling processes essential for survival and
development [29 31]. The detoxification of reactive
oxygen species and alkyl hydroperoxides appears espe-
cially important for parasitic organisms which have to
cope not only with their metabolically produced oxygen
radicals but also with those generated by the hostimmune system. In case of the malaria parasite P.
falciparum reactive oxygen species are formed during
the catabolism of host cell haemoglobin and generate
an enhanced oxidative stress on parasite and host cell
which needs to be combatted [32 34]. Apart from
enzymes such as catalase and glutathione peroxidase it
is very likely that the thioredoxin redox cycle consisting
of NADPH dependent thioredoxin reductase, thiore-
doxin and thioredoxin dependent peroxidases supplies
an additional antioxidative system to protect P. falci-
parum from oxidative damage. Thioredoxin reductase
was recently identified in the parasites but it was not
certain until now whether the parasites possess a func-
tional thioredoxin redox system [19]. Here we report on
the identification of the gene for P. falciparum thiore-
doxin and the recombinant expression and characterisa-
tion of the parasite protein.
The nucleotide sequence of PfTrx was identified in
the TIGR database on chromosome 14. The coding
sequence is interrupted by one intron. Interestingly, the
thioredoxin reductase gene is located on the same chro-
mosome as the gene for glutathione reductase. One
could speculate that the transcription of these relatedgenes is correlated according to the needs of the para-
site. However, since the sequencing and assembly of
tion period. Incubation of 250 mM DTNB without
addition of protein resulted in the release of 9.6 mM
TNB. The reaction of PfTrxC30A with DTNB
yielded in the release of 1.63 equivalents of TNB,
suggesting that in this mutant protein Cys33 is fully
accessible for modification with TNB and that the
additional 0.63 equivalents are due to the modification
of Cys43 (as in the wild-type protein). Interestingly, the
situation is different in PfTrxC33A, where only 0.86
equivalents of TNB are released. Attributing about
0.7 equivalents to a modification of Cys43, then Cys30
seems to be buried in the protein structure. These datasuggest, that Cys33 rather than Cys30 is the thiol which
interacts with PfTrxR during the reduction process and
is also responsible for the transfer of electrons to the
acceptor molecule.
3.4. Kinetic properties of P. falciparum thioredoxin
reductase with thioredoxin
The steady state kinetic parameters of the reaction of
PfTrxR with PfTrx and PfTrxC43A are summarized in
Table 2. The Km-values determined were in the range of0.82.1 mM and the specific activity of PfTrxR with
PfTrx or PfTrxC43A was determined to be 1238
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Z. Krnajski et al. /Molecular & Biochemical Parasitology 112 (2001) 219228224
chromosome 14 is still in progress it remains for later
analyses to address this question.
The coding region of PfTrx consists of 312 nucle-
otides and encodes for a polypeptide of 104 amino
acids. The deduced amino acid sequence contains the
typical thioredoxin CysGlyProCys active site motif and
shows the highest degree of identity to thioredoxin II
from S. cere6isiae and S. pombe, respectively. This
implies that the malaria parasite most likely possesses
more than one thioredoxin like almost all other organ-
isms investigated so far [3538]. The increasing number
of plant thioredoxins which are present in distinct
forms in cytosol and mitochondria and their functional
analyses suggest that different thioredoxins reduce pre-
ferred substrates like distinct protein disulphides or
reactive oxygen species [36,38]. Some of the functions
Fig. 4. Absorption spectra of PfTrxWT and mutant proteins before and after modification with DTNB. (A) 50 mM PfTrxWT before modification
with 250 mM DTNB (line a) and the fraction containing the highest amount of TNB-modified PfTrxWT after gel filtration on Sephadex G-25 (line
b); (B) 50 mM PfTrxC30A before modification with 250 mM DTNB (line a) and the fraction containing the highest amount of TNB-modified
PfTrxC30A after gel geltration on Sephadex G-25 (line b); (C) 50 mM PfTrxC33A before modification with 250 mM DTNB [line a] and the
fraction containing the highest amount of modified PfTrxC33A after gelfiltration on Sephadex G-25 (line b); (D) 50 mM PfTrxC43A before (linea) and after modification with DTNB (line b). Modification of all proteins except PfTrxC43A leads to the formation of a new absorbance band
around 324 nm.
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Table 1
Modification of P. falciparum thioredoxin wild-type and mutant
proteins with DTNBa
TNB [mM]Protein species modified with [TNB]/[Trx]
DTNB
0.71PfTrxWT 35.391.1
1.6381.599.5PfTrxC30A
43.594.7PfTrxC33A 0.86
0.15PfTrxC43A 7.590.5
a Fifty micromolar of recombinant proteins were reacted with
5-fold molar excess of DTNB overnight and separated from non-re-
acted DTNB and released TNB by gel filtration on Sephadex G-25.
All fractions were analysed spectrophotometrically and the concen-
tration of TNB released was calculated using the molar extinction
coefficient 13 600 M1 cm1 at 412 nm for the anion. As a control
250 mM DTNB was incubated overnight without addition of protein
and the release of TNB was determined to be 9.690.15 mM.
Fig. 5. Steady state kinetic analyses of PfTrxR with PfTrx without
and with addition of PfTrxC30A or PfTrxC33A. PfTrxR activity
was assayed in the coupled insulin test with increasing concentrations
of PfTrx (0.58 mM) without () or with addition of 5 mM
PfTrxC30A () or with addition of 5 mM PfTrxC33A ().
of the thioredoxin system can be fulfilled by the glu-
tathione reductase/glutathione/glutaredoxin redox cycleas has been demonstrated in S. cere6isiae and E. coli
[39 41]. These observations suggest that the mainte-
nance of an adequate redox milieu and the detoxifica-
tion of reactive oxygen species is essential in aerobic
organisms since several backup systems occur in one
cell. Even in trypanosomatids two systems exist. Until
recently it was thought that these organisms only pos-
sess the trypanothione dependent redox cycle [42 44]
but Krauth-Siegel and co-workers [45] have isolated a
thioredoxin from Trypanosoma brucei and Myler et al.
[46] have reported of a thioredoxin-related sequencelocated on Leishmania major chromosome 1. P. falci-
parum also possess a functional glutathione system in
addition to the thioredoxin system [18,4750].
Purified PfTrxWT and mutant proteins precipitated
readily which may be due to the occurrence of a high
number of hydrophobic residues in addition to a third
cysteine residue (Cys43) in the primary structure of
PfTrx, which may confer dimer formation. Mutation of
Cys43 into alanine resulted in a protein species that
precipitated less than PfTrxWT. On SDS-PAGE the
small portion of dimers observed could not be reversed
by b-mercaptoethanol, whereas the reductant had a
strong effect on the dimerization of the wild-type
protein. The tendency to form dimers was also found in
the thioredoxin recombinantly expressed from T. brucei
and Cys67, the only cysteine residue in addition to the
active site cysteines, was suggested to be responsible for
this interaction [45]. Human thioredoxin contains three
additional cysteine residues which are responsible for
aggregation of the protein [51]. This process has been
suggested to autoregulate availability and activity in
vivo [52]. In cancerous tissues it has been shown that
thioredoxin is overexpressed and most likely reaches
concentrations where dimers are the predominant form
of the protein [53,54]. There is a variety of hypotheses
about the biological role of this dimer formation suchas the inhibition of normal thioredoxin activities by
elimination of redox function or acquisition of a spe-
cific activity unique to the dimer form [51].
Steady state kinetic analyses of PfTrxWT and mu-
tant proteins with PfTrxR showed that PfTrxWT and
PfTrxC43A are almost equally well accepted as sub-
strates by PfTrxR, whereas PfTrxC30A and
PfTrxC33A are not substrates for the reductase, as
expected. Comparison with E. coli thioredoxin which
was used as a model substrate for PfTrxR in the past
demonstrates that the reduction of PfTrx is about250-fold more efficient than for the model substrate. A
certain degree of specificity for their endogenous sub-
Table 2
Steady state kinetic parameters of PfTrxR with PfTrxWT,
PfTrxC43A and E.coli Trx
Substrate kcat/Kmkcat min1Km [mM]
16749196 7912.190.2PfTrxWTa
PfTrxWTb 4391.490.4 6069135
PfTrxWTc 11669130 6331.891.1
PfTrxC43Aa 1685950 13231.390.2
754616964PfTrxC43Ab 0.890.1
PfTrxC43Ac 20389199 21531.090.2
16889153500925 3E. coli Trxa
a Thioredoxin reductase/thioredoxin assay coupled with insulin (see
Section 2).b Thioredoxin reductase/thioredoxin assay coupled with DTNB
(see Section 2).c Thioredoxin reductase/thioredoxin assay coupled with thiore-
doxin peroxidase 1 of P. falciparum (see Section 2).
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Z. Krnajski et al. /Molecular & Biochemical Parasitology 112 (2001) 219228226
strates was also found for TrxRs from mammals. For
instance the affinity of the rat liver enzyme for the
natural thioredoxin is about 10-fold higher than for E.
coli thioredoxin [55]. Mammalian TrxRs reduce a num-
ber of substrates which are not reduced by the parasite
protein. It is likely that the extraordinary occurrence of
the C-terminal cysteine-selenocysteine pair in the mam-
malian protein is responsible for this wide substratespecificity [56 58]. PfTrxR possesses a CysGlyGlyG-
lyLysCys motif at the C-terminus which has been
shown to be involved in catalysis [21,22,59]. Chemically
there are considerable differences between a sulphur
and a selenium and the redox active residues are sepa-
rated by four amino acid residues in the parasite
protein whereas they are adjacent in the mammalian
enzyme. These distinct features give hope for the iden-
tification of compounds that specifically target the par-
asite enzyme.
It was shown that human TrxR is competitively
inhibited by active-site mutants of human thioredoxin
[60] whereas the mutant proteins PfTrxC30A and
PfTrxC33A at 5 mM did not inhibit PfTrxR.
The accessibility of the active site thiols of PfTrx was
analysed using DTNB and the mutant proteinsPfTrxC30A and PfTrxC33A. According to our results,
Cys33 is accessible for modification with the com-
pound, whereas Cys30 is buried in the protein. In E.coli and human thioredoxins the cysteine residue equiv-
alent to Cys30 in PfTrx is the one which is most
accessible whereas the second cysteine is buried in the
structure of the protein [61]. Our data suggest that thestructure of P. falciparum thioredoxin is different from
those of the well investigated E. coli and human
proteins and it remains for further investigation to
elucidate the precise topology and the mechanism of
action of PfTrx with PfTrxR and its acceptor
molecules. In mammalian and E. coli thioredoxins the
mechanisms of action for thioredoxin as a protein
disulphide reductase is based on an initial nucleophilic
attack by the thiolate of Cys32 (equivalent to Cys30 in
PfTrx) with the formation of an unstable transient
mixed disulphide involving Cys32 and one of the sul-
furs in the substrate. This is followed by a conforma-
tional change and a nucleophilic attack of Cys35
(equivalent to Cys33 in PfTrx) to reform the disulphide
between Cys32 and 35 [61].
The identification of PfTrx offers excellent possibili-
ties to elucidate the precise biological functions of the
thioredoxin system for the development and survival of
the malaria parasite P. falciparum. One possible func-
tion of the thioredoxin system in the parasite is the
removal of hydrogen peroxide by thioredoxin depen-
dent peroxidases. One of these proteins from P. falci-
parum was recombinantly expressed and shown toaccept reducing equivalents from PfTrx and to transfer
them to hydrogen peroxide (Muller et al., unpublished
data). This peroxiredoxin possesses one potential active
site cysteine residue and surprisingly it accepts reducing
equivalents from thioredoxin in contrast to other 1-Cys
peroxiredoxins which are reported to use a different
unidentified source as electron donor in other systems
[12].
Further, the evaluation and assessment of PfTrxR as
a potential target for chemotherapy of malaria appearsto be more feasible when using the natural substrate of
the enzyme in inhibitor studies rather than having to
use model substrates which may interact in a different
way with PfTrxR. For example, it has been suggested
that DTNB which is one of the model substrates forPfTrxR, interacts primarily with the N-terminal redox
active cysteine centre of the protein since removal of
the C-terminal cysteine residues had only a moderate
effect on DTNB reduction [21].
Acknowledgements
The authors like to thank B. Bergmann for excellent
technical assistance. This research is supported by a
grant of the Deutsche Forschungsgemeinschaft (MU
837/11). This article is part of a doctoral study at the
University of Hamburg, Faculty of Biology (Z.K.).
S.M. is a Wellcome Senior Research Fellow in Basic
Biomedical Science.
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