In most living cells, redox homeostasis is based both on the glutathione and the thioredoxin system. In the malaria parasite Plasmodium falciparum antioxidative proteins represent promising targets for the development of antiparasitic drugs. We cloned and expressed a thioredoxin of P. falciparum (pftrx), and we improved the stable expression of the thioredoxin reductase (PfTrxR) of the parasite by multiple silent mutagenesis. Both proteins were biochemically characterized and compared with the human host thioredoxin system. Intriguingly, the 13-kDa protein PfTrx is a better substrate for human TrxR (K m ‫؍‬ 2 M, k cat ‫؍‬ 3300 min ؊1 ) than for P. falciparum TrxR (K m ‫؍‬ 10.4 M, k cat ‫؍‬ 3100 min ؊1 ). Possessing a midpoint potential of ؊270 mV, PfTrx was found to reduce the disease-related metabolites S-nitrosoglutathione and GSSG. The rate constant k 2 for the reaction between reduced P. falciparum thioredoxin and GSSG was determined to be 0.039 M ؊1 min ؊1 at 25°C and pH 7.4. The k 2 for thioredoxins from man, Drosophila melanogaster, and Escherichia coli was ϳ5 times lower. Our data suggest that GSSG reduction can be supported at a high rate by the TrxR/Trx system in glutathione reductase-deficient cells; this may be relevant for certain stages of the malarial parasite but also for cells containing high [GSSG] of other organisms like dormant forms of Neurospora, glutathione reductasedeficient yeast mutants, or CD4 ؉ lymphocytes of AIDS patients.

The glyoxalase system consisting of glyoxalase I (GloI) and glyoxalase II (GloII) constitutes a glutathione-dependent intracellular pathway converting toxic 2-oxoaldehydes, such as methylglyoxal, to the corresponding 2-hydroxyacids. Here we describe a complete glyoxalase system in the malarial parasite Plasmodium falciparum. The biochemical, kinetic and structural properties of cytosolic GloI (cGloI) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) were directly compared with the respective isofunctional host enzymes. cGloI and cGloII exhibit lower K(m) values and higher catalytic efficiencies (k(cat)/K(m) ) than the human counterparts, pointing to the importance of the system in malarial parasites. A Tyr185Phe mutant of cGloII shows a 2.5-fold increase in K(m) , proving the contribution of Tyr185 to substrate binding. Molecular models suggest very similar active sites/metal binding sites of parasite and host cell enzymes. However, a fourth protein, which has highest similarities to GloI, was found to be unique for malarial parasites; it is likely to act in the apicoplast, and has as yet undefined substrate specificity. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were active in the lower nanomolar range. The Glo system of Plasmodium will be further evaluated as a target for the development of antimalarial drugs.

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development.Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 M )1 AEs)1 at pH 6.9 and 25°C; glutathione-dependent reduction of H 2 O 2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites.Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.

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Recombinant Plasmodium falciparum glyoxalase I (PfGlx I) was characterized as monomeric Zn 2+ -containing enzyme of 44 kDa. The K M value of the methylglyoxal^gluta-thione adduct is 77 þ 15 W WM, the k cat value being 4000 min 31 at 25 ‡C and pH 7.0. PfGlx I consists of two halves, each of which is homologous to the small 2-domain glyoxalase I of man. Both parts of the pfglx I gene were overexpressed; the C-terminal half of PfGlx I was found to be a stable protein and formed an enzymatically active dimer. These results support the hypothesis of domain-swapping and subunit fusion as mechanisms in glyoxalase I evolution.