o parasita da malária plasmodium falciparum possui um funcional

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: [email protected] (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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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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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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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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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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o parasita da malária plasmodium falciparum possui um funcional

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