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Marcel Deponte

Publications and source records attributed to Marcel Deponte.

9 recordsLinked to original sources
PubMed2005

Plasmodium falciparum glutathione S-transferase--structural and mechanistic studies on ligand binding and enzyme inhibition.

Glutathione S-transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high- and a low-affinity binding site. This is supported by a clear uncompetitive inhibition type. Site-directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH-dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 A resolution X-ray structure of PfGST cocrystallized with the inhibitor S-hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C-terminal end of helix alpha4 and at the S-hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.

Animals↗
PubMed2005

Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase.

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.

Binding Sites↗
PubMed2005

Biochemical characterization of Toxoplasma gondii 1-Cys peroxiredoxin 2 with mechanistic similarities to typical 2-Cys Prx.

TgPrx2 represents a recently discovered cytosolic 1-Cys peroxiredoxin (Prx) from the intracellular parasite Toxoplasma gondii. Over-expression of the respective gene confers protection against H(2)O(2), suggesting that the protein possesses peroxidase activity. According to the current nomenclature eukaryotic typical and atypical 2-Cys Prx contain a second conserved resolving cysteine residue whereas 1-Cys Prx work on the basis of a monothiol mechanism. Only a few 1-Cys peroxiredoxins have been biochemically characterized to date. Here we describe the mechanistic characterization of TgPrx2 in vitro, including site directed mutagenesis studies, gel filtration chromatography, and molecular modeling. TgPrx2 has general antioxidant properties as indicated by its ability to protect glutamine synthetase against a dithiothreitol Fe(3+)-catalyzed oxidation system. However, TgPrx2 does not reduce H(2)O(2) nor tert-butyl hydroperoxide at the expense of glutaredoxin, thioredoxin or glutathione. Cys(47) was identified as the active site cysteine residue. Most interestingly, Cys(47) was found to form an intermolecular disulfide with Cys(209) from the C-terminal domain of a second subunit which acts as the resolving cysteine. This is a mechanism analogous to typical peroxiredoxins. In contrast to the latter, however, dimeric TgPrx2 does not oligomerize to decamers but is able to form tetramers and hexamers which are non-covalently associated. To our knowledge, TgPrx2 is the first eukaryotic 'so called' 1-Cys peroxiredoxin shown to act on the basis of a 2-Cys mechanism. Our data indicate that mechanistic studies are essential for classifying peroxiredoxins.

Amino Acid Sequence↗
PubMed2005

Glutathione S-transferase from malarial parasites: structural and functional aspects.

Malaria represents an emerging disease because of increasing parasite resistance against available drugs and because of increasing geographical distribution of the causative agent, Plasmodium falciparum. The complete genome of Plasmodium was sequenced recently, revealing that the parasite harbors only one glutathione S-transferase (PfGST). This observation was of particular interest: First, certain antimalarial drugs such as chloroquine and methylene blue presumably influence the glutathione metabolism in which PfGST is involved. Second, PfGST might play a significant role in drug resistance. PfGST was studied in parasite extracts and as recombinant protein, and its x-ray structure has been solved. The available data indicate that the homodimeric PfGST cannot be assigned to any of the previously known GST classes. PfGST exhibits significant structural differences to human GSTs, particularly at the so-called hydrophobic binding pocket (H-site) where the second substrate binds. Inhibition of PfGST is expected to act at different vulnerable metabolic sites of the parasite in parallel; it is likely to disturb GSH-dependent detoxification processes, to increase the levels of cytotoxic peroxides, and possibly to increase the concentration of toxic hemin. In this chapter, we summarize the current knowledge on PfGST, including aspects of structure, function, and future drug development.

Animals↗
PubMed2005

Plasmodium falciparum glutaredoxin-like proteins.

Glutaredoxin-like proteins form a new subgroup of glutaredoxins with a serine replacing the second cysteine in the CxxC-motif of the active site. Yeast Grx5 is the only glutaredoxin-like protein studied biochemically so far. We identified and cloned three genes encoding glutaredoxin-like proteins from the malaria parasite Plasmodium falciparum (Pf Glp1, Pf Glp2, and Pf Glp3) containing a conserved cysteine in the CGFS-, CKFS-, and CKYS-motif, respectively. Here, we describe biochemical properties of Pf Glp1 and Pf Glp2. Cys 99, the only cysteine residue in Pf Glp1, has a pK(a) value as low as 5.5 and is able to mediate covalent homodimerization. Monomeric and dimeric Pf Glp1 react with GSSG and GSH, respectively. Pf Glp2 is monomeric and both of its cysteine residues can be glutathionylated. Molecular models reveal a thioredoxin fold for the putative C-terminal domain of Pf Glp1, Pf Glp2, and Pf Glp3, as well as conserved residues presumably required for glutathione binding. However, Pf Glp1 and Pf Glp2 neither possess activity in a classical glutaredoxin assay nor display activity as glutathione peroxidase or glutathione S-transferase. Mutation of Ser 102 in the CGFS-motif of Pf Glp1 to cysteine did not generate glutaredoxin activity either. We conclude that, despite their ability to react with glutathione, glutaredoxin-like proteins are a mechanistically and functionally heterogeneous group with only little similarities to canonical glutaredoxins.

Amino Acid Sequence↗
PubMed2005

Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts.

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.

Amino Acid Sequence↗
PubMed2005

Plasmodium falciparum 2-Cys peroxiredoxin reacts with plasmoredoxin and peroxynitrite.

Thioredoxin peroxidase 1 (TPx1) of the malarial parasite Plasmodium falciparum is a 2-Cys peroxiredoxin involved in the detoxification of reactive oxygen species and - as shown here - of reactive nitrogen species. As novel electron acceptor of reduced TPx1, we characterised peroxynitrite; the rate constant for ONOO- reduction by the enzyme (1 x 10(6) M(-1) s(-1) at pH 7.4 and 37 degrees C) was determined by stopped-flow measurements. As reducing substrate of TPx1, we identified - aside from thioredoxin - plasmoredoxin; this 22-kDa protein occurs only in malarial parasites. When studying the potential roles of Cys74 and Cys170 of Tpx1 in catalysis, as well as in oligomerisation behaviour, we found that replacement of Cys74 by Ala influenced neither the dimerisation nor enzymatic activity of TPx1. In the C170A mutant, however, the kcat/Km for reduced Trx as a substrate was shown to be approximately 50-fold lower and, in contrast to the wild-type enzyme, covalently linked dimers were not formed. For the catalytic cycle of TPx1, we conclude that oxidation of the peroxidatic Cys50 by the oxidising substrate is followed by the formation of an intermolecular disulfide bond between Cys50 and Cys170' of the second subunit, which is then attacked by an external electron donor such as thioredoxin or plasmoredoxin.

Amino Acid Substitution↗
PubMed2003

Plasmodium falciparum thioredoxins and glutaredoxins as central players in redox metabolism.

Over the last few years, an increasing number of different functions have been ascribed to small redox-active proteins like thioredoxins (Trx) and glutaredoxins (Grx). These functions include redox regulation of transcription and translation, antioxidant defence, involvement in protein folding and cellular signalling, and reduction of ribonucleotide reductase. In the malarial parasite Plasmodium falciparum, a classical Trx and a typical Grx have been described as well as a number of Trx- and Grx-like proteins including monothiol glutaredoxins. Furthermore, plasmoredoxin, a redox-active protein related to Trx, has been characterized; plasmoredoxin is unique for malarial parasites, therefore having great potential as diagnostic tool. In this minireview, we summarize the current knowledge on members of the thioredoxin superfamily and their function in the malarial parasite P. falciparum.

Amino Acid Sequence↗