Molecular & Biochemical Parasitology 112 (2001) 219 228 www.parasitology-online.

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The malaria parasite Plasmodium falciparum possesses a functional thioredoxin 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 0.8 2.1 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 efciency of PfTrxR to reduce the isolated PfTrx is at least 250-fold higher than to reduce E. coli thioredoxin. PfTrx contains a 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 modication by this compound. Surprisingly the rst cysteine residue of the active site motif (Cys30) is less accessible than the second cysteine (Cys33), which is highly prone to the modication. 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

1. Introduction The thioredoxin system consists of the NADPH dependent disulphide oxidoreductase thioredoxin reducAbbre6iations: DTNB, 5,5%-dithiobis (2-nitrobenzoic acid); DTT, dithiothreitol; PfTrx, P. falciparum thioredoxin; PfTrxR, P. falciparum thioredoxin reductase; TNB; 5%-thionitrobenzoic acid. * Corresponding author. Present address: Department of Biochemistry, 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 available 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, Australia.

tase (TrxR) and the small protein thioredoxin. This system is responsible for several redox reactions within the 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 reductase and methionine sulfoxide reductase [14]. In addition it has been shown that thioredoxins are involved in transcriptional control by modifying the redox state of thiols in the active site of transcription factors and altering their activation state. Transcription factors regulated by thioredoxins are OxyR, NF-kB

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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and AP-1 [59]. 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 peroxiredoxins. This increasing family of proteins has been identied in a wide variety of organisms and its abundance in the cell has led to the suggestion that it represents one of the major peroxide detoxifying proteins [10 12]. It certainly plays an important role in helminths where it was suggested to be the key enzymatic 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 efciency for the reduction of hydrogen peroxide [14 16] and the thioredoxin system is proposed to be the main detoxication system for reactive oxygen species. It is well established that Plasmodium infected erythrocytes are under enhanced oxidative stress. However, the parasite-host cell unit appears to be able to maintain the necessary balance between oxidants and antioxidants so that parasite development is not impaired. Several enzymatic antioxidants have been isolated from the parasites [14,17,18] but their role for parasite survival awaits evaluation. We have isolated and partially characterised TrxR from P. falciparum [19 22] and have now identied a thioredoxin-like sequence 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 the reduction 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 library of P. falciparum was a kind gift from Professor David Kaslow, Bethesda, USA. Bovine insulin and 5,5%-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma. NADPH was from Boehringer Mannheim. Recombinant TrxR from P. falciparum was prepared as described in Gilberger et al. [21].

Research website (www.tigr.org). Sequencing of chromosome 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 specic sense oligonucleotide 5%GCGCGCATATGGTAAAAATTGTAACTAGTC-3% coding for the rst seven amino acids of the potential P. falciparum thioredoxin and the antisense oligonucleotide 5%-GCGCGCTCGAGTTAAGCTGCGTATTTTTCG-3% encoding the last six amino acids of the potential coding region of the genomic sequence, a 315 bp fragment was amplied 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 thioredoxin coding region was amplied using Pfu polymerase (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 puried, 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 ml 1 ampicillin was inoculated. The bacterial culture was diluted 1:100 into Terric Broth containing 50 mg ml 1 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 temperature 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 Tris HCl buffer pH 7.9 containing 100 mM NaCl, 40 mM imidazol and 1 mM dithiothreitol (DTT) and stored at 20C. The protein was puried using Ni2 + -chelating chromatography according to the manufacturers recommendation (Qiagen). Protein concentration was calculated by using the molar extinction coefcient of 13 700 M 1 cm 1 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 sense 5%-GCTGAATGGGCTGGACCATGCAAAAG-3% and antisense 5%-CTTTTGCATGGTCCAGCCCATTCA-

2.2. Isolation of a P. falciparum thioredoxin-like sequence and expression of the recombinant protein
Preliminary sequence data for P. falciparum chromosome 14 was obtained from The Institute for Genomic

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GC-3% to replace Cys30 by alanine and sense 5%-GAATGGTGTGGACCAGCCAAAAGAATTGCCCC-3% and antisense 5%-GGGGCAATTCTTTTGGCTGGTCCACACCATTC-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 formation of the wild-type protein. The mutagenic oligonucleotides were: sense 5%-CCCATTTTATGAAGAAGCCTCCAAAACATACAC-3% and antisense 5%-GTGTATGTTTTGGAGGCTTCTTCATAAAATGGG-3%. All mutations were veried by nucleotide sequencing [24]. The plasmids (pJC40) containing the mutated open reading frames were transformed into E. coli BL 21 (DE3) and expression and purication was performed 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 kinetic parameters were compared. The insulin assay mixture contained 100 mM Hepes pH 7.6, 0.2 mM EDTA, 200 mM NADPH, 1 10 mM PfTrx or PfTrxC43A and 2 mg ml 1 insulin and the change in absorbance at 340 nm was determined. The DTNB assay mixture contained essentially the same components as described above but instead of insulin, 40 mM DTNB 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 expressed potential thioredoxin peroxidase (peroxiredoxin) 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 ml 1 P. falciparum peroxiredoxin (Muller et al., unpublished data). The Km-values, turnover numbers and catalytic efciencies for PfTrxR and the respective substrates under the different assay conditions were determined in duplicate using 35 independent protein preparations. Standard 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.

present in PfTrx (Cys30, 33 and 43) are accessible to solvent, PfTrxWT and mutant proteins were modied 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 mM EDTA 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 calculated by using the molar extinction coefcient of 13 600 M 1 cm 1 at 412 nm for TNB. After modication the proteins were separated from non-reacted DTNB and free TNB by gel ltration 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-modied proteins and free TNB were identied by absorption spectrophotometry (240 580 nm).

3. Results

3.1. Sequence analysis

Using PCR the coding region of the putative PfTrx was amplied. The deduced amino acid sequence encodes 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 contains 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 thioredoxin [24 26]. In addition a third cysteine residue was identied in position 43 which may be involved in the formation of protein dimers in vitro and in vivo (Fig. 2).

3.2. Expression and purication of recombinant P. falciparum thioredoxin

PfTrx wild-type and mutant proteins were expressed in E. coli BL 21 (DE3) as His-tagged fusion proteins

2.5. Thiol accessibility

To investigate whether all three cysteine residues

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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 purication by Ni2 + -chelating chromatography. During the purication 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 purication was largely diminished by constant precipitation of the proteins. According to gel ltration 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 purication procedure and that these dimers are partly attributable to the formation of intermolecular disulphide bridges (Fig. 2).

spectroscopy (240 580 nm) and the concentration of released TNB was calculated (Table 1). DTNB treatment with PfTrxWT results in the release of 0.71 M equivalents of TNB, suggesting that there is one accessible thiol in this protein species. According to the deduced amino acid sequence we assume that this reaction is attributable to modication 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-

3.3. Modication of P. falciparum thioredoxin wild-type and mutant proteins with DTNB
PfTrxWT, PfTrxC30A, PfTrxC33A and PfTrxC43A were modied with DTNB which resulted in the formation 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 coefcient of 13 600 M 1 cm 1 for TNB [27]. PfTrxWT, PfTrxC30A, PfTrxC33A and PfTrxC43A exhibit symmetrical absorption peaks at 280 nm whereas the modied proteins PfTrxWT, PfTrxC30TNB and PfTrxC33-TNB develop pronounced shoulders around 324 nm, respectively (Fig. 4 A D). PfTrxRC43A shows no modication after treatment with DTNB because there are no free thiols accessible in this protein species (Fig. 4 D). To obtain full modication the proteins were incubated with DTNB overnight and then the modied protein species were separated from excess DTNB and formed TNB by gel ltration. All fractions were analysed by absorption

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 kDa ladder. Proteins were resolved on a 15% SDS-PAGE and visualized with coomassie brilliant blue.

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units mg 1 depending on the assay performed. These differences are possibly due to the different ability of PfTrx to interact with different acceptor molecules used in the assays. Obviously DTNB represents a poor acceptor for the reducing equivalents of PfTrx or PfTrxC43A, although it has been reported to interact perfectly well with E. coli thioredoxin [28]. In comparison E. coli thioredoxin was used as a substrate for PftrxR in the insulin assay and the Km-value determined was 500 mM with a kcat of 1688 min 1. 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 PfTrxR PfTrx reaction, but the reduction was not impaired by the mutant proteins (Fig. 5).

4. Discussion
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 00 000 g supernatant of E. coli BL21 (DE3) containing the expression plasmid of PfTrxWT (3 mg). Lane 2: 1 mg of PfTrxWT after purication with Ni2 + -chelating chromatography. Lane 3: 1 mg of PfTrxC30A after purication with Ni2 + -chelating chromatography. Lane 4: 1 mg of PfTrxC33A after purication with Ni2 + -chelating chromatography. Proteins were resolved on a 15% SDS-PAGE and visualized with coomassie brilliant blue.

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 modication with TNB and that the additional 0.63 equivalents are due to the modication 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 modication of Cys43, then Cys30 seems to be buried in the protein structure. These data suggest, 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 of 0.8 2.1 mM and the specic activity of PfTrxR with PfTrx or PfTrxC43A was determined to be 12 38

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 control and signalling processes essential for survival and development [29 31]. The detoxication of reactive oxygen species and alkyl hydroperoxides appears especially important for parasitic organisms which have to cope not only with their metabolically produced oxygen radicals but also with those generated by the host immune 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, thioredoxin and thioredoxin dependent peroxidases supplies an additional antioxidative system to protect P. falciparum from oxidative damage. Thioredoxin reductase was recently identied in the parasites but it was not certain until now whether the parasites possess a functional thioredoxin redox system [19]. Here we report on the identication of the gene for P. falciparum thioredoxin and the recombinant expression and characterisation of the parasite protein. The nucleotide sequence of PfTrx was identied 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 chromosome as the gene for glutathione reductase. One could speculate that the transcription of these related genes is correlated according to the needs of the parasite. However, since the sequencing and assembly of

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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 nucleotides 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 organisms investigated so far [35 38]. 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 preferred 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 modication with DTNB. (A) 50 mM PfTrxWT before modication with 250 mM DTNB (line a) and the fraction containing the highest amount of TNB-modied PfTrxWT after gel ltration on Sephadex G-25 (line b); (B) 50 mM PfTrxC30A before modication with 250 mM DTNB (line a) and the fraction containing the highest amount of TNB-modied PfTrxC30A after gel geltration on Sephadex G-25 (line b); (C) 50 mM PfTrxC33A before modication with 250 mM DTNB [line a] and the fraction containing the highest amount of modied PfTrxC33A after gelltration on Sephadex G-25 (line b); (D) 50 mM PfTrxC43A before (line a) and after modication with DTNB (line b). Modication of all proteins except PfTrxC43A leads to the formation of a new absorbance band around 324 nm.

Z. Krnajski et al. / Molecular & Biochemical Parasitology 112 (2001) 219228 Table 1 Modication of P. falciparum thioredoxin wild-type and mutant proteins with DTNBa Protein species modied with DTNB PfTrxWT PfTrxC30A PfTrxC33A PfTrxC43A
a

225

TNB [mM]

[TNB]/[Trx]

35.3 9 1.1 81.59 9.5 43.59 4.7 7.5 9 0.5

0.71 1.63 0.86 0.15

Fifty micromolar of recombinant proteins were reacted with 5-fold molar excess of DTNB overnight and separated from non-reacted DTNB and released TNB by gel ltration on Sephadex G-25. All fractions were analysed spectrophotometrically and the concentration of TNB released was calculated using the molar extinction coefcient 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 9.6 9 0.15 mM.

of the thioredoxin system can be fullled by the glutathione reductase/glutathione/glutaredoxin redox cycle as has been demonstrated in S. cere6isiae and E. coli [39 41]. These observations suggest that the maintenance of an adequate redox milieu and the detoxication 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 possess 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 sequence located on Leishmania major chromosome 1. P. falciparum also possess a functional glutathione system in addition to the thioredoxin system [18,47 50]. Puried 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
Table 2 Steady state kinetic parameters of PfTrxR with PfTrxWT, PfTrxC43A and E.coli Trx Substrate PfTrxWTa PfTrxWTb PfTrxWTc PfTrxC43Aa PfTrxC43Ab PfTrxC43Ac E. coli Trxa Km [mM] 2.1 90.2 1.4 9 0.4 1.8 9 1.1 1.3 9 0.2 0.8 9 0.1 1.0 9 0.2 500 9 25 kcat min1 16749 196 606 9135 11669 130 16859 50 6169 64 20389 199 16889153 kcat/Km 791 439 633 1323 754 2153 3

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 (0.5 8 mM) without (
) or with addition of 5 mM PfTrxC30A ( ) or with addition of 5 mM PfTrxC33A ( ).

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 thioredoxin peroxidase 1 of P. falciparum (see Section 2).

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 such as the inhibition of normal thioredoxin activities by elimination of redox function or acquisition of a specic activity unique to the dimer form [51]. Steady state kinetic analyses of PfTrxWT and mutant proteins with PfTrxR showed that PfTrxWT and PfTrxC43A are almost equally well accepted as substrates 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 about 250-fold more efcient than for the model substrate. A certain degree of specicity for their endogenous sub-

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strates was also found for TrxRs from mammals. For instance the afnity of the rat liver enzyme for the natural thioredoxin is about 10-fold higher than for E. coli thioredoxin [55]. Mammalian TrxRs reduce a number 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 mammalian protein is responsible for this wide substrate specicity [56 58]. PfTrxR possesses a CysGlyGlyGlyLysCys 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 separated by four amino acid residues in the parasite protein whereas they are adjacent in the mammalian enzyme. These distinct features give hope for the identication of compounds that specically target the parasite 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 proteins PfTrxC30A and PfTrxC33A. According to our results, Cys33 is accessible for modication with the compound, whereas Cys30 is buried in the protein. In E. coli and human thioredoxins the cysteine residue equivalent 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 the structure 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 sulfurs in the substrate. This is followed by a conformational change and a nucleophilic attack of Cys35 (equivalent to Cys33 in PfTrx) to reform the disulphide between Cys32 and 35 [61]. The identication of PfTrx offers excellent possibilities to elucidate the precise biological functions of the thioredoxin system for the development and survival of the malaria parasite P. falciparum. One possible function of the thioredoxin system in the parasite is the removal of hydrogen peroxide by thioredoxin dependent peroxidases. One of these proteins from P. falciparum was recombinantly expressed and shown to accept 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 unidentied source as electron donor in other systems [12]. Further, the evaluation and assessment of PfTrxR as a potential target for chemotherapy of malaria appears to 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 for PfTrxR, 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 837/1 1). 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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