Eur. J. Biochem. 268, 1404±1409 (2001) q FEBS 2001
Thioredoxin peroxidases of the malarial parasite Plasmodium falciparum Stefan Rahlfs and Katja Becker Interdisciplinary Research Center, Justus-Liebig-University, Giessen, Germany
The open reading frames of two different proteins with homologies to 2-Cys peroxiredoxins have been identified in the P. falciparum genome. Both genes, with a length of 585 and 648 bp, respectively, were amplified from a gametocyte cDNA and overexpressed in Escherichia coli. The gene products (deduced m 21.8 and 24.6 kDa) with an overall identity of 51.8% were found to be active in the glutamine synthetase protector assay. The smaller protein (named Pf-thioredoxin peroxidase 1; PfTPx1) is reduced by P. falciparum thioredoxin (PfTrx) and accepts H2O2, t-butylhydroperoxide, and cumene hydroperoxide as substrates, the respective kcat values for the N-terminally
His-tagged protein in the presence of 10 mm PfTrx and 200 mm substrate being 67, 56, and 41 min21 at 25 8C. As described for many peroxiredoxins, PfTPx1 does not follow saturation kinetics. Furthermore, in oxidizing milieu both proteins are converted to another protein species migrating faster in SDS gel electrophoresis. For PfTPx1 also this second species was found to be active, however, with different kinetic properties which might indicate a mechanism of enzyme regulation in vivo.
Peroxiredoxins form a recently discovered and ubiquitously distributed family of antioxidant enzymes that act as peroxidases by reducing hydrogen peroxide and alkyl hydroperoxides to water or the corresponding alcohol [1±3]. In contrast to many other peroxidases, the function of peroxiredoxins does not depend on redox cofactors such as metals or prosthetic groups [4]. The peroxiredoxin superfamily, which also includes glutathione peroxidases and tryparedoxin peroxidase [5], can be divided into two subgroups, the 1-Cys peroxiredoxin and the 2-Cys peroxiredoxin, depending on the presence of one or two conserved cysteine-containing motifs [1±3]. 2-Cys peroxiredoxin use electrons provided by the small protein thioredoxin and were thus also named thioredoxin peroxidases (TPx; formerly called thiol-specific antioxidants) [2,6]. Oxidized thioredoxin in turn is reduced by thioredoxin reductase (TrxR), an NADPH-dependent flavoenzyme [7]. As the first exception to this rule, a 1-Cys peroxiredoxin with thioredoxin peroxidase activity has recently been reported in yeast [8]. Antioxidant defense plays a vital role in the malarial parasite P. falciparum. This is due to the rapid multiplication of blood stage parasites in an environment of high oxygen tension [9] but also to the fact that the parasites digest large quantities of hemoglobin and therefore have to detoxify prooxidative heme iron [10]. This susceptibility of malarial parasites is employed for new approaches to rational drug design.
Recently we demonstrated the presence of a functional thioredoxin system in P. falciparum. This system consists of the homodimeric flavoenzyme thioredoxin reductase [11] and a typical thioredoxin [12]. The sequence of a glutathione peroxidase-like peroxiredoxin has been demonstrated in the genome of the parasites by Gamain and coauthors [13] although the complete kinetic characterization of this protein has not yet been published. A 1-Cys peroxiredoxin has been cloned and characterized in P. falciparum [14]. Whether this protein is thioredoxin dependent or not is presently under investigation, however, to be studied. Here we report on the cloning, heterologous expression, and first biochemical and kinetic characterization of two different 2-Cys peroxiredoxins from P. falciparum.
Correspondence to K. Becker, Interdisciplinary Research Center, Giessen University, Heinrich-Buff-Ring 26±32, 35392 Giessen, Germany. Fax: 1 49 6419939129, Tel.: 1 49 641 9939120, E-mail:
[email protected] Abbreviations: TPx, thioredoxin peroxidase; TrxR, thioredoxin reductase; Trx, thioredoxin. (Received 10 November 2000, revised 3 January 2001, accepted 9 January 2001)
Keywords: antioxidant defense; malaria; peroxiredoxin; P. falciparum; thioredoxin system.
M AT E R I A L S A N D M E T H O D S Materials All chemicals used were of the highest available purity and were obtained from Roth or Merck; t-butylhydroperoxide was from Fluka and cumene hydroperoxide from Sigma. The cloning vector pBluescript SK1 was obtained from Stratagene, the expression system QIAexpress (vector pQE30, Escherichia coli host strain M15, and nickelnitriloacetic acid matrices for purification of His-tagged protein) was purchased from Qiagen. PCR-primers were obtained from MWG-Biotech, the sequencing reactions were carried out by MWG-Biotech. PfTrxR and PfTrx were recombinantly produced and purified as described previously [12]. E. coli glutamine synthetase was purchased from Sigma-Aldrich. Phosphatidylcholine hydroperoxide was kindly provided by Prof. Brigelius-FloheÂ, Deutsches Institut fuÈr ErnaÈhringsforschung, Potsdam. PCR amplification, sequencing, and subcloning Complete open reading frames of two different 2-Cys peroxiredoxin-like genes were identified by online screening
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of the P. falciparum genome sequencing project on chromosome 12 and 14, respectively (www.ncbi.nlm.nih. gov/Malaria/plasmodiumblcus.html). Neither of the two genes contains introns. Two sets of two homologous primers were derived from the genes. For subsequent cloning procedures, restriction site sequences (underlined) were introduced for BamHI and HindIII at the 5 0 end of the respective primer (for chromosome 14: N-terminal primer: OPf14tpx5 0 : 5 0 -CGCGGGATCCGCATCATATGTAGGAAGAGAAGC-3 0 ; C-terminal primer: OPf14tpx3 0 : 5 0 -GCGCAAGCTTTTACAACTTTGATAAATATTCAC-3 0 ; for chromosome 12: N-terminal primer: OPf12tpx5 0 : 5 0 -CGCGGGATCCTTTTTAAAAAAACTGTGCAGGAGC-3 0 ; C-terminal primer: OPf12tpx3 0 : 5 0 -GCGCAAGCTTTTAA gametocyte TACATTTTTATTTGCATTATTC-3 0 ). cDNA-library obtained from the P. falciparum strain 3D7 was kindly provided by D. Kaslow (NIH, Bethseda, MD, USA) [15] and used as a template to amplify the two open reading frames by PCR (2.5 min 94 8C; 94 8C, 30 s; 58 8C, 30 s; 72 8C, 60 s; 25 cycles; 72 8C, 2 min). The derived fragments of correct size were cloned into pBluescript SK1 for sequencing and for subcloning into the expression vector pQE30. Gene expression and purification of the recombinant proteins The E. coli strain M15 was used for expression of the two P. falciparum peroxiredoxin genes. Competent cells were transformed with the respective pQE30/ Tpx plasmid. Cells were grown at 37 8C in Luria±Bertani medium containing ampicillin (100 mg´mL21) and kanamycin (50 mg´mL21) to a density where D600 0.5; subsequently the expression was induced by adding 1 mm isopropyl thio-b-d-galactoside. Cells were grown for additional 4 h, harvested and directly used for protein-purification or frozen at 220 8C. For purification, the cells were disintegrated by sonication in the presence of protease inhibitors. After centrifugation the supernatant was loaded onto a nickel-nitriloacetic acid column equilibrated with 50 mm sodium phosphate, 300 mm NaCl, pH 8.0. After washing the column with increasing imidazole concentrations, the proteins were eluted with 175±200 mm imidazol; collected fractions were tested for enzyme activity and for purity by 12% SDS/PAGE. Active fractions were pooled and concentrated via ultrafiltration and dialyzed against 50 mm Hepes, pH 7.2 prior to use. Protein concentrations were determined by the Bio-Rad dye assay with BSA as a standard and on the basis of the calculated extinction coefficient of the respective protein (:calc. 280 nm for His-tagged recombinant protein: 21.71 mm21´cm21 for PfTPx1 and 21.80 mm21´cm21 for the second 2-Cys peroxiredoxin (P. falciparum peroxiredoxin 2, which will be referred to from here on as `putative PfTPx2'). Determination of antioxidant activity of PfTPx The overall antioxidant activity of PfTPx1 and the putative PfTPx2 was estimated by measuring the activity to protect the inactivation of E. coli glutamine synthetase by a thiol metal-catalyzed oxidation system (dithiothreitol/Fe31/O2; thiol mixed-function oxidase system [4]); with modifications. Briefly, a 50-mL reaction mixture containing
glutamine synthetase, 10 mm dithiothreitol, 8 mm FeCl3, 50 mm Hepes buffer, pH 7.2, and various concentrations of reduced protector protein (PfTPx1 or putative PfTPx2) or control proteins (BSA, PfTrx) was incubated for 15 min at 30 8C. Subsequently, 0.5 mL `glutamine synthetase assay mixture' (400 mm ADP, 150 mm glutamine, 10 mm potassium arsenate, 20 mm NH2OH, 400 mm MnCl2 in 100 mm Hepes, pH 7.4) was added and incubated for another 20 min at 37 8C before the reaction was stopped with 0.5 mL stopsolution (55 g FeCl3´6H2O, 20 g trichloroacetic acid, and 21 mL concentrated HCl per L). The absorbance at 540 nm was taken as a measure for the glutamine synthetasecatalyzed reaction. Under these conditions glutamine synthetase was inhibited (in the absence of protector protein) by 82%. This reaction could partially be prevented by the addition of 2 mm EDTA (20% residual inhibition). Thioredoxin peroxidase assay TPx-assays were carried out at 25 8C in an assay mixture of 1 mL consisting of 50 mm Hepes, pH 7.2, with 100 mm NADPH, 50 mU PfTrxR (as determined with PfTrx as substrate; care was taken that the PfTrxR concentration did not become rate limiting), 10 mm PfTrx, and 200 mm substrate (hydroperoxide (H2O2), t-butylhydroperoxide, cumene hydroperoxide, or phosphatidylcholine hydroperoxide. For further kinetic characterization, PfTrx and substrate concentrations were systematically varied. The assay was started with TPx; NADPH consumption (:340 6.22 mm21´cm21) was monitored spectrophotometrically at 340 nm. In this coupled assay system constantly high concentrations of reduced thioredoxin were maintained by the NADPH/ TrxR system.
R E S U LT S A N D D I S C U S S I O N Cloning and sequencing of the P. falciparum 2-Cys peroxiredoxin genes Screening of the P. falciparum genome sequencing database resulted in the finding of two P. falciparum sequences with high homologies to known TPx sequences. Primers were designed and PCRs were performed with PfcDNA as template. Two fragments with expected size were obtained and cloned into the vector pSK1. Nucleotide sequences were determined for both DNA strands of the respective PCR products. The amino-acid sequences were in full agreement with the respective genomic database sequences, despite one conservative T!C mismatch in the DNA sequence of tpx1 at position 150 of the gene. This exchange results in a TGC-triplet instead of TGT, both coding for cysteine. The Pftpx1-gene (GenBank accession no. AF225977) is located on chromosome 14 and comprises 585 bp, putative Pftpx2 (GenBank accession no. AF225978) is on chromosome 12 and comprises 648 bp. Overexpression of the two recombinant P. falciparum peroxiredoxin genes and purification of the respective proteins The two tpx genes were each inserted into the expression vector pQE30. Freshly transformed E. coli M15 cells were used for production of recombinant P. falciparum TPxs.
1406 S. Rahlfs and K. Becker (Eur. J. Biochem. 268)
Fig. 1. 12% SDS/PAGE of PfTPx1 freshly purified in the presence of 0.5 mm dithiothreitol (lane 4) and without dithiothreitol (lane 3). Lane 1 shows PfTPx1 after 1 week at 4 8C without dithiothreitol treatment. The putative PfTPx2 (purified in the presence of dithiothreitol) is shown on lane 5. Lane 2 contains the molecular mass marker with sizes shown on the right.
The N-terminal hexahistidyl-tag contributed by the pQE vector allowed easy purification over nickel-nitriloacetic acid agarose columns in the presence (, 1 mm) or absence of dithiothreitol. All assays described in this paper were carried out with the His-tagged proteins, the characteristics of which might differ slightly from the wild-type enzymes. According to silver stained SDS-gel electrophoresis both proteins were . 99% pure. The calculated molecular masses (of the His-tagged proteins) are 22.9 kDa for PfTPx1 and 25.8 kDa for the putative PfTPx2. These values correspond well to the data obtained by SDS/PAGE (Fig. 1). Further characteristics of the two proteins are summarized in Table 1. In the presence of 1 mm dithiothreitol, the concentrated proteins were stable at 4 8C for a week. Longer storage, particularly in the absence of reducing agents, may lead to the appearance of the second protein species (described below) and loss of activity.
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Fig. 2. Alignment of the amino-acid sequence of the two P. falciparum (Pf ) thioredoxin peroxidases with thioredoxin peroxidase sequences of man (hs), Mus musculus (Mm), Schizosaccharomyces pombe (Sp), and Schistosoma mansoni (Sm). The active site motifs are indicated by boxes.
Antioxidant properties of P. falciparum thioredoxin peroxidases The deduced amino-acid sequences of both Pftpx-genes contain two active site `VCP' motifs that are characteristic for 2-Cys peroxiredoxin or TPx [1,2,16] (see Fig. 2). The two P. falciparum proteins share 51.8% sequence identity. Concerning other species, both proteins show highest homologies with proposed or known 2-Cys peroxiredoxins from plants such as Hordeum vulgare, Triticum aestivum, Secale cereale, and Spinacea oleracea. This fact further supports the putative `green origin' of Plasmodium. The rather long N-terminal sequence (see Fig. 2) of the putative PfTPx2 was tested in a signal peptide prediction algorithm (http://www.cbs.dtu.dk/services/ TargetP/) [17]. Interestingly, there is a 91.5% prediction for a mitochondrial
Table 1. Properties of thioredoxin peroxidases of P. falciparum. ND, not determined.
GenBank acc. no. Location Size of the ORF, including start codon (bp) Amino acids m (kDa) a IEP a :280 (mm21´cm21) a Conserved Cys in N-terminal region Conserved Cys in C-terminal region Additional Cys residues Dependency pH Optimum a
Calculated, without His-tag;
b
PfTPx1
Putative PfTPx2
AF225977 Chromosome 14 585
AF225978 Chromosome 12 648
195 21.8 7.2 21.7 Cys50 Cys179 Cys74 PfTrx (Km 4 mm) 7.2
216 24.7 8.7 21.8 Cys67 Cys187 Cys7, 54, 55, 152 Thioredoxin? ND
determined in the presence of 200 mm H2O2.
b
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Thioredoxin peroxidases of P. falciparum (Eur. J. Biochem. 268) 1407
Table 2. Kinetic properties of thioredoxin peroxidase 1 of P. falciparum. ND, not determined. For the determination of apparent Km values the activity obtained at 200 mm peroxide substrate was defined to equal Vmax; the reaction catalyzed by PfTPx1 does not follow saturation kinetics. The activity was determined in the presence of 10 mm PfTrx and 200 mm H2O2 at 25 8C. 50% of maximal activity (mm) measured using Substrate
5 mm PfTrx
10 mm PfTrx
20 mm PfTrx
Specific activity (U´mg21)
H2O2 t-Butylhydroperoxide Cumene hydroperoxide
9.4 ND ND
16 4 2
41 8.6 11
3.1 2.6 1.9
targeting sequence with a proposed cleavage site at residue 19 [18]. In our experiments, the recombinant protein produced contained this putative mitochondrial targeting sequence. This could explain the fact that no adequate peroxidase activity was detectable. This aspect is presently under further investigation. PfTPx1 and the putative PfTPx2 were tested in the glutamine synthetase inactivation assay described by Kim et al. [4]. In this assay system, glutamine synthetase is incubated with FeCl3 and dithiothreitol in the presence and absence of antioxidant proteins. This leads to an oxidative inactivation of glutamine synthetase which is (partially) prevented by the protector protein tested. Under our conditions, the maximal inhibition achieved by the ironcatalyzed inactivation resulted in 18% residual glutamine synthetase activity. The presence of 2 mm EDTA led to a reduction of enzyme inhibition resulting in 80% residual activity. The addition of 10 mm freshly produced prereduced PfTPx1 to the initial reaction mixture of 50 mL led to 86% enzyme activity indicating strong antioxidant activity of the protein. The addition of 10 mm putative PfTPx2 had only a slight protective effect and will be re-evaluated after removal of the mitochondrial targeting sequence. As controls, 10 mm BSA and 10 mm PfTrx, respectively, were included and did not result in any protection. These data show that PfTPx1 can act as sulfur radical scavenger, which is a characteristic feature of thioredoxin peroxidases [4]. Kinetic characterization of PfTPx1 In a second step, the recombinant peroxiredoxins were studied for peroxidase activity. In the presence of NADPH and PfTrxR (as a thioredoxin regenerating system) as well as Pf-thioredoxin, PfTPx1 efficiently catalyzes the reduction of H2O2, t-butylhydroperoxide, and cumene hydroperoxide. At 25 8C and in the presence of 10 mm PfTrx and 200 mm substrate the determined specific activities are 3.1, 2.6, and 1.9 U´mg21, respectively. This result demonstrates that the protein is indeed a thioredoxin-dependent peroxidase. Therefore we named the protein PfTPx1. Very recently, a 1-Cys peroxiredoxin has been described in P. falciparum by Kawazu et al. [14]. This protein was, however, found to be glutathione dependent and an activity with thioredoxin has not been demonstrated. As described for many other peroxiredoxins including glutathione peroxidases and tryparedoxin peroxidase [5,19] the reaction catalyzed by PfTPx1 does not follow typical saturation kinetics at high substrate concentrations. Under
kcat (min21) 67 56 41
these conditions, definite Vmax and Km values cannot be determined [5,19]. Furthermore, when approximating Km values, different values were obtained when varying the PfTrx concentration. As an example, when determining an apparent Km in a Lineweaver±Burk plot in the presence of 4 mm to 200 mm H2O2, a Km value of 10 mm was determined in the presence of 5 mm PfTrx, a value of 17 mm was determined in the presence of 10 mm PfTrx, and 41 mm in the presence of 20 mm PfTrx (see Table 2). These data indicate that the underlying catalytic mechanism of the enzyme is complex but might be described e.g. by the special Dalziel equation for bisubstrate reactions [20]. The Km for PfTrx in the presence of 200 mm H2O2 was estimated to be 4 mm. A detailed kinetic characterization of PfTPx1 is presently being carried out. Using hydrogen peroxide as substrate, a pH-profile of PfTPx1 in 50 mm Hepes indicated an optimum at pH 7.2. The addition of potassium chloride to final concentrations of 50, 100, and 200 mm, respectively, slightly decreased the enzyme activity. The putative PfTPx2 did not show significant activity under the conditions described above. The presence of six cysteine residues in the protein which are prone to intramolecular and intermolecular reactions might contribute to this lack of activity. Furthermore, neither PfTPx1 nor the putative PfTPx2 were found to be glutathione dependent or catalyzed reduction of the hydrophobic substrate phosphatidylcholine hydroperoxide. For measuring kinetic parameters on the two peroxiredoxins, enzyme fractions were used that had been purified in the presence of 500 mm dithiothreitol. Dithiothreitol was then removed by dialysis directly prior to use of the proteins. Interestingly, when purified without dithiothreitol for both proteins a second band appeared on SDS-gels which was about 2 kDa smaller. The same phenomenon was observed in dithiothreitol-containing samples that had been stored for . 1 week. This protein species did not disappear by treatment with 100 mm dithiothreitol for 12 h; it might be generated by an intramolecular reaction or, less likely, by partial degradation of the protein [6,21]. As the hexahistidyl-affinity-tag had been attached to the N-terminal end of the protein, a degradation, which takes already place prior to or during purification, would have to affect the C-terminus. Also, for human erythrocyte plasma membrane TPx a second, smaller band has been reported. This protein species was found to be the result of a cleavage of a C-terminal fragment; both cysteine-motifs as well as peroxidase activity were still present [21]. For a mammalian
1408 S. Rahlfs and K. Becker (Eur. J. Biochem. 268)
1-Cys peroxiredoxin, however, the upper and lower band could be reversibly converted into each other by the addition or absence of dithiothreitol [6]. As shown for PfTPx1, the protein species representing the lower band in the SDS gel electrophoresis is also reduced by PfTrx and turns over H2O2, t-butylhydroperoxide, and cumene hydroperoxide. An enzyme sample containing only the lower band was obtained by purifying the recombinant protein in the absence of dithiothreitol and storing it at 4 8C until the upper band had completely disappeared. However, the pH optimum of the reaction is shifted from 7.2 to 8.0 and the approximated Km values (at fixed substrate concentrations, see above) increase by a factor of 5. As this, probably oxidized, conformation of PfTPx1 is very stable and still active, it might be part of a regulatory process which could be of relevance in vivo. This aspect is currently being studied in more detail. Antioxidant defense plays a crucial role in the malarial parasite P. falciparum. Thus, the redox metabolism of the parasites represents an important target for current and future anti-malarial strategies [22]. Peroxiredoxins, as major peroxide-detoxifying systems, are therefore of particular interest. Until now a glutathione peroxidase-like protein has been described in P. falciparum [13]. The substrate specificity of this protein is presently under investigation [23]. Furthermore, a 1-Cys peroxiredoxin of P. falciparum has recently been described [14]. Whether this enzyme depends on thioredoxin has, however, not yet been investigated. According to our data, P. falciparum possesses at least one typical and highly active thioredoxin peroxidase named PfTPx1. The second peroxiredoxin described here shows all sequence characteristics of a TPx; the substrate specificity of this protein has, however, not yet been elucidated. Our future studies will focus on further kinetic and structural characterization of the two 2-Cys peroxiredoxins as well as on their stage-specific expression and intraparasitic distribution.
ACKNOWLEDGEMENTS The technical assistance of Marina Fischer and Petra Harwaldt is highly acknowledged. The study was supported by the Deutsche Forschungsgemeinschaft (SFB 544/535 and Be 1540/4-1). Sequence data for P. falciparum chromosome 12 was obtained from the Stanford Genome Technology Center website at http://www-sequence.stanford. edu/group/malaria. Sequencing of P. falciparum chromosome 12 was accomplished as part of the Malaria Genome Project with support by the Burroughs Wellcome Fund. Preliminary sequence data for P. falciparum chromosome 14 was obtained from The Institute for Genomic 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 US Department of Defense.
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