Thioredoxin and glutathione system of malaria parasite Plasmodium falciparum.

Plasmodium falciparum is the causative agent of malaria tropica. Due to the increasing resistance towards the commonly used plasmodicidal drugs there is an urgent need to identify and assess new targets for the chemotherapeutic intervention of parasite development in the human host. It is established that P. falciparum-infected erythrocytes are vulnerable to oxidative stress, and therefore efficient antioxidative systems are required to ensure parasite development within the host cell. The thioredoxin and glutathione redox systems represent two powerful means to detoxify reactive oxygen species and this article summarizes some of the recent work which has led to a better understanding of these systems in the parasite and will help to assess them as potential targets for the development of new chemotherapeutics of malaria.

The malaria parasite Plasmodium falciparum possesses a functional thioredoxin system.

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 11715 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 K(m)-values for PfTrx in the range of 0.8--2.1 microM 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 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 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.

The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs.

The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.

Intersubunit interactions in Plasmodium falciparum thioredoxin reductase.

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M(r) and the low M(r) proteins. The well characterized Escherichia coli TrxR represents a member of the low M(r) class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M(r) proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M(r) TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol.

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.

The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.-W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584-29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C-terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant P/TrxRC535AC540A and the deletion mutant PfTrxRdelta9 (C-terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5'-dithiobis (2-nitrobenzoate). These data imply that the C-terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.

Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell. Recent studies revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases. The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. The putative essential amino acids at the catalytic center of large thioredoxin reductase from P. falciparum were determined by using site-directed mutagenesis techniques. To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues. Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine. All mutants were expressed in the E. coli system and characterized. Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity. These results clearly identify Cys88 and Cys93 as the active site thiols of large thioredoxin reductase. The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity. These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase. Spectral analysis of wild-type P. falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase. This spectral feature, recently also reported for the human placenta protein (Arscott, L. D., Gromer, S., Schirmer, R. H., Becker K., and Williams, C. H., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3621-3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to small E. coli thioredoxin reductase.

Molecular characterization and expression of Onchocerca volvulus glutathione reductase.

Glutathione metabolism represents a potential target for anti-parasite drug design. The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The gene is composed of 13 exons and 12 introns and spans 4065 bp. The first intron is located within the 5'-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed. The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5' end, 236 nucleotides upstream of the first in-frame methionine. The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR). A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR). The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 microM) over NADH (Km 108 microM). The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.