112,714 research outputs found
A plant-type enzyme as a putative target for novel antimalarial drugs : properties of the Plasmodium falciparum ferredoxin-NADP+ reductase
No full textApicomplexan parasites harbor a specific organelle, named apicoplast, which is related to plant non-photosynthetic plastids and displays a plant-like metabolism. The apicoplast has been shown to contain typical vegetal proteins, such as ferredoxin–NADP+ reductase (FNR) and ferredoxin (Fd) (1-2). Both proteins from Plasmodium falciparum (PfFNR and PfFd) have been produced in recombinant form and characterized (3). The PfFNR/PfFd couple was shown to be catalytically active in vitro yielding reducing power to support the activity of LytB (4), the last enzyme of the biosynthetic pathway for isoprenoid precursors, a known site of action of antiplasmodial compounds. On this basis, PfFNR has been proposed as a possible target for new antimalarial drugs (2).
The three-dimensional structure of PfFNR has been determined by X-ray crystallography (3). Compared to other plastidic-type FNRs, PfFNR displays a significantly lower catalytic efficiency and lower selectivity against NADH. These functional features are probably the consequence of the lack of protein positively-charges stabilizing the 2’-phosphate of the bound substrate. NADP(H) binding to PfFNR occurs through an induced-fit mechanism never observed in other FNRs. The conformational changes induced by binding to the enzyme of 2’-P-AMP, a NADP+ analogue, includes the partial unwinding of an α-helix localized in the NADP+-binding domain. Furthermore, the binding of NADP+ triggers the formation of a disulfide-stabilized homodimer resulting in the inactivation of PfFNR. This process, observed in vitro, could represent a physiologic mechanism regulating the enzyme activity.
Structure-based design of PfFNR inhibitors is in progress and has already yielded some active compounds, with inhibitory constants in the range of micromolar or lower.
1. Pandini V. et al. (2002) J. Biol. Chem. 277, 48463-48471
2. Seeber F. et al. (2005) Curr. Farm. Des. 11, 3159-7312
3. Milani et al. (2007) J. Mol. Biol. 367, 501-513
4. Röhrich R.C. et al. (2005) FEBS lett. 579, 6433-643
Structure-function studies of the major insertion of the apicomplexan ferredoxin NADP+ reductase, investigated by mutagenesis and limited proteolysis
No full textApicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical and economic importance. They include the human pathogens Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, an opportunistic human parasite. It was shown that most members of this phylum harbor a plastid-like organelle, called the apicoplast, of vegetal origin. The plant-type ferredoxin/ferredoxin-NADP+ reductase (Fd/FNR) redox system found in this organelle has been proposed as a target for novel drugs (1, 2). Elucidation of the properties and functions of this redox system would be greatly facilitated by a more detailed structural knowledge of the two components. Like many proteins from these protists, apicomplexan FNRs are characterized by the presence of unique peptide insertions of variable length and yet unknown function. The major insertion of T. gondii FNR (TgFNR) (28 aa with respect to maize root FNR) is near the FAD isoalloxazine ring binding motif. Thus, it is expected that it could influence the protein-protein interaction of the redox couple.
The aim of this study was therefore to investigate the structure and the involvement of this region of TgFNR in substrate binding by mutagenesis and limited proteolysis approaches.
All four proteinases tested yielded a limited number of peptides. Most of the cleavage sites were located within the major insertion of TgFNR, indicating that this region is surface exposed and is highly flexible. Furthermore, protection of the insertion by TgFd against proteolytic cleavage indicates a role for this region in ferredoxin binding (3).
Three different deleted forms of the parasite enzyme were produced, in which the insertion was fully removed (Del1), partially removed (Del2) or replaced with the homologous region of plant FNR (Del3). Compared to the wild type enzyme, Del2 and Del3 showed similar values for the kinetic parameters of the TgFd-dependent NADPH-cytochrome c reductase activity, while Km for ferredoxin of Del1 was highly increased. By affinity chromatography on immobilized TgFd, deleted TgFNRs were shown to bind more weakly TgFd than the wild-type enzyme. Del1 showed a remarkable loss of affinity for ferredoxin, probably because it lacks three positively charged residues which are present in Del2 and in Del3 mutants.
The results reported here allow us to conclude that the insertion of TgFNR is solvent exposed and structurally flexible. It is suggested to give rise to a 38-residue intrinsically unstructured subdomain, protruding from the top of the FAD-binding beta-barrel. Such a species-specific subdomain does not have major functions in protein folding and stability. Rather, it participates in TgFd binding, significantly increasing the stability of the protein-protein complex and improving the catalytic efficiency of the enzyme in the electron transfer to TgFd.
1. M. Vollmer et al. (2001) J. Biol. Chem. 276(8): 5483-5490
2. V. Pandini et al. (2002) J Biol Chem. 277(50):48463-48471.
3. V. Pandini et al. (2006) Biochemistry 45: 3563-357
New insights on the molecular mechanism of action of human renalase
No full textRenalase is a new human flavoprotein possibly involved in blood pressure and cardiac function regulation1. Despite the potential implications for human health, the biochemical properties of renalase have not been investigated so far, so that the molecular mechanism underlying its action is far from been understood. The low sequence identity with MAOs led to the hypothesis that renalase could represents a new class of monoamine oxidases, acting by degrading plasma catecholamines2,3. In order to elucidate the mechanism of action of human renalase at a molecular level, three recombinant forms of human renalase were produced in E. coli yielding amounts of protein suitable for its biochemical characterization4. Spectroscopic analyses revealed for the first time that renalase is a flavoprotein, with FAD as noncovalently-bound cofactor. At variance with MAOs, recombinant renalase was found to be devoid of any oxidase activity toward biogenic amines. Nevertheless, its unusual reactivity towards various compounds sheds a new light on the nature of the reaction catalyzed by this enzyme.
1. Xu J. et al. (2005) J. Clin. Invest. 115, 1275-1280
2. Li G. et al. (2008) Circulation 117, 1277-1282
3. Wang J. et al. (2008) Mol. Biol. Rep. 35, 613-620
4. Pandini V. et al. (2010) Protein Expr. Purif. 72, 244-5
Ferredoxin–NADP+ reductases of Apicomplexa: unique properties of protozoan plant–type enzymes
No full textPlant-type ferredoxin-NADP+ reductases (FNRs) are a family of flavin-dependent dehydrogenases/electron transferases ubiquitous in plant plastids, cyanobacteria and some eubacteria (1). Recently, FNRs have been identified in the apicoplast of apicomplexan parasites, which includes the causative agents of malaria and toxoplasmosis. FNRs from Toxoplasma gondii (TgFNR) and Plasmodium falciparum (PfFNR) have been cloned and characterized (2, 3). Whereas TgFNR failed to yield crystals suitable for X-ray analysis, the crystal structure of PfFNR has been determined (3). Compared to FNRs from other sources, PfFNR displays a significantly lower catalytic efficiency and poorer NADH/NADPH selectivity (3). These features of PfFNR have been interpreted on the basis of the peculiar structure of its NADP(H)-binding site. Furthermore, PfFNR undergoes an unprecedented NADP-triggered, redox-dependent homodimerization process leading to enzyme inactivation, which could represent a physiologic mechanism of enzyme regulation. PfFNR has been shown to be involved in the biosynthesis of isoprenoid precursors (4), which is the site of action of known antiplasmodial compounds. On this basis, apicomplexan FNRs have been proposed as a possible new target for novel antiparasitic drugs (2).
References
1. Aliverti, A., Pandini, V., Pennati, A., de Rosa, M., and Zanetti, G., Arch. Biochem. Biophys., 474, 283-291 (2008).
2. Seeber, F., Aliverti, A., and Zanetti, G., Curr. Farm. Des., 11, 3159-7312 (2005).
3. Milani, M., Balconi, E., Aliverti, A., Mastrangelo, E., Seeber, F., Bolognesi, M., and Zanetti, G., J. Mol. Biol., 367, 501-513 (2007).
4. Röhrich, R.C., et al., FEBS Lett., 579, 6433–6438 (2005)
Functional characterization of Plasmodium falciparum ferredoxin-NADP+ reductase
No full textApicomplexa are protozoan parasites responsible for infective diseases of major worldwide impact, like malaria or toxoplasmosis. We have identified and characterized a redox system of Toxoplasma gondii (1), comprising ferredoxin-NADP+ reductase and ferredoxin, which is of vegetal origin and has not a homolog in the human host, thus being possibly a novel drug target (2). This system is localized in the apicoplast, an organelle shown to be essential for parasite survival both in P. falciparum and T. gondii. Recently, we have demonstrated the ability of the redox system FNR/Fd of P. falciparum to reconstitute in vitro the electron transfer pathway to the enzyme LytB which catalyzes the last step of the mevalonate-independent isoprenoid biosynthesis in the apicoplast (3). Here, we report a thorough characterization of the ferredoxin-NADP+ reductase of P. falciparum (PfFNR). We have cloned and overproduced in Escherichia coli the PfFNR in soluble and active form. The recombinant PfFNR, purified to homogeneity, has been studied with respect to the spectral properties and the interaction with its protein partner P. falciparum ferredoxin. Steady-state and rapid kinetics studies show several differences between the P. falciparum redox system and those of both plants and T.gondii. Redox titration experiments indicate that redox potential of the FAD prosthetic group of PfFNR is by far more positive than that of photosynthetic FNRs. Inhibition studies with organic and transition metal salts are in progress as well as the screening of conditions to obtain crystals for X-ray structure analysis.
1 Pandini V., Caprini G., Thomsen N., Aliverti A., Seeber F., Zanetti G. (2002) J. Biol. Chem. 277, 48463-48471.
2 Seeber F., Aliverti A., Zanetti G. (2005) Curr. Pharm. Des. 11, 3159-3172.
3 Rohrich RC, Englert N, Troschke K, Reichenberg A, Hintz M, Seeber F, Balconi E, Aliverti A, Zanetti G, Kohler U, Pfeiffer M, Beck E, Jomaa H, Wiesner J. (2005) FEBS Lett. 579,6433-8
Towards the molecular mechanism of action of human renalase on the cardiovascular system
No full textINTRODUCTION: Within a post-genomic survey of human genes encoding soluble, potentially secreted proteins of unknown function, a putative enzyme carrying a FAD-binding signature was identified (1). This protein, which displays low similarity to flavin-containing monoamine oxidases (MAOs) was named renalase, since it is preferentially synthesized in the kidney (1). It has been proposed to be secreted into the bloodstream, where it was shown to modulate blood pressure and heart rate (1, 2). It was hypothesized that renalase could represent a novel type of MAO, acting on circulating catecholamines (1, 3). Thus, renalase may be a novel key participant in the interplay between the excretory and circulating systems. Low blood renalase level in patients suffering from chronic kidney disease might be responsible for the increased risk of cardiovascular complications in these subjects (4). With the aim to clarify the mechanism of action of human renalase at the molecular level, we have undertaken the purification and characterization of the protein produced in Escherichia coli.
RESULTS: Recombinant human renalase has been produced in E. coli as a FAD-containing soluble protein and purified (5), allowing the determination of its biochemical properties, which unexpectedly were markedly different from those of mammalian MAOs. The protein is a monomer containing no disulfide bridge. The cofactor is firmly, but not covalently bound to the apoprotein and its reactivity towards different compounds is at variance with those of MAOs. Finally, recombinant renalase was found to be devoid of any oxidase activity towards various biogenic amines. On the contrary, we found experimental evidence that renalase could catalyze reactions different from oxidative deamination. We conclude that the previous proposal that renalase is a novel MAO isoform should be rejected.
1. Xu J. et al. (2005) J. Clin. Invest. 115, 1275-1280
2. Li, G. et al. (2008) Circulation 117, 1277-1282
3. Wang J. et al. (2008) Mol. Biol. Rep. 35, 613-620
4. Desir G. V. (2009) Kidney Int. 76, 366-370
5. Pandini V. et al. (2010) Protein Expr Purif. doi:10.1016/j.pep.2010.03.00
Interactive effects between carbon allotropes on the mechanical reinforcement of nanocomposites based on poly(1,4- cis -isoprene)
No full textInteractive effects of carbon allotropes on the mechanical reinforcement of polymer nanocomposites were investigated by using, as the polymer matrix, poly(1,4-cis-isoprene) (PI) samples from industrial synthesis and from natural sources. Carbon nanotubes (CNT) and nano-graphite with high shape anisotropy (nanoG) were melt blended with PI, as the only fillers or in combination with carbon black (CB), measuring the shear modulus at low strain amplitudes. The nanofiller was found to increase the low amplitude storage modulus of the matrix, with or without CB, by a factor depending on nanofiller type and content. The filler-polymer interfacial area was able to correlate modulus data of composites with CNT, CB and with the hybrid filler system, leading to the construction of a common master curve. The filler networking phenomenon was found to be affected by type and amount of low molecular mass products of PI from natural sources. The correlation between chemical composition, dynamic mechanical and ultimate properties of nanocomposites was investigated. In particular, it was found that low molecular mass components control the ability of elastomeric nano-composites to store or dissipate energy
Plasmodium falciparum ferredoxin-NADP+ reductase : a plant-type enzyme as a promising new target for novel antimalarial drugs
No full textINTRODUCTION: Plant-type ferredoxin-NADP+ reductases (FNRs) are a widespread group of flavin-dependent enzymes that catalyze the exchange of reducing equivalents between NADP(H) and ferredoxin (Fd) (1). FNRs have been found in plant plastids, cyanobacteria and some eubacteria (1), where they form electron transport chains involved in biosynthetic processes as diverse as photosynthesis, nitrogen assimilation, and response against reactive oxygen species. FNRs have been recently identified in the apicoplast of apicomplexan parasites (2, 3), which includes the causative agents of malaria and toxoplasmosis. Plasmodium falciparum FNR (PfFNR) and Fd (PfFd) have been cloned and functionally characterized (4-6). The PfFNR/PfFd couple has been shown to support in vitro the activity of LytB (6), the last enzyme of the biosynthetic pathway for isoprenoid precursors, which is the site of action of known antiplasmodial compounds. On this basis, PfFNR has been proposed as a possible new target for antimalarial drugs (3).
RESULTS: The structure of PfFNR has been determined by X-ray crystallography. Compared to other plastidic-type FNRs, PfFNR displays a significantly lower catalytic efficiency and lower selectivity against NADH. These functional features are probably the consequence of the lack of protein positively-charged groups stabilizing the 2’-phosphate of bound substrate. PfFNR interacts with the adenine moiety of the bound NADP(H) through a His residues not conserved in other FNRs. The role of this residue in catalysis has been investigated by site-directed mutagenesis. NADP(H)-binding to PfFNR occurs through an induced-fit mechanism unprecedented in other enzyme of this protein family. The conformational changes induced by binding to the enzyme of 2’-P-AMP, a NADP analogue, include the partial disruption of an α-helix localized in the NADP-binding domain. Furthermore, PfFNR was shown to undergo NADP+-triggered homodimerization in vitro, resulting in the formation of an intermolecular disulfide bridge and leading to enzyme inactivation. This process, which can be fully reversed by cleaving the disulfide by reductants like DTT or lipoate, could represent a physiologic mechanism regulating the enzyme activity. Structure-based design of PfFNR inhibitors is in progress and has already yielded some active compounds.
1. Ceccarelli E.A. et al. (2004) Biochim. Biophys. Acta 1698, 155-165
2. Pandini V. et al. (2002) J. Biol. Chem. 277, 48463-48471
3. Seeber F. et al. (2005) Curr. Farm. Des. 11, 3159-7312
4. Milani M. et al. (2007) J. Mol. Biol. 367, 501-513
5. Kimata–Ariga Y. et al. (2007) J. Biochem. 142, 715-720
6. Röhrich R.C. et al. (2005) FEBS Lett. 579, 6433–643
Plasmodium falciparum ferredoxin-NADP+ reductase : induced-fit transition in the NADP-binding domain and ligand-triggered dimerization and inactivation
No full textNew antimalarial drugs are urgently needed to tackle the spread of Plasmodium falciparum strains resistant to current pharmacological treatments. The recent completion of the genome sequence of this parasite (1) has considerably boosted the search of new drug targets. Plasmodia, as most protists belonging to the phylum Apicomplexa, possess a nonphotosyntetic plastid, dubbed the apicoplast (2), which is essential for parasite survival and has no counterpart in the human host (3). We have shown that the apicoplast of both T. gondii and P. falciparum contains a short plant-type electron transport chain, comprising ferredoxin NADP+ reductase (FNR) and ferredoxin (Fd) (4,5). Furthermore, we have demonstrated that such enzyme system is able to provide electrons to the enzyme LytB, which catalyzes the last step of the mevalonate-independent isoprenoid biosynthesis in the apicoplast (6). We have cloned and overproduced in E. coli both P. falciparum FNR (PfFNR) and Fd (PfFd). The two proteins have been purified, characterized and shown to be catalytically competent as an NADPH-dependent electron transfer system, though displaying specific functional features that distinguish them from their plant homologs. Recently, we have obtained the crystal structures of PfFNR both in free form and in complex with the substrate analog 2P-AMP, at 2.4 and 2.7 Å resolution, respectively. Surprisingly, PfFNR displayed structural properties unique to the P. falciparum enzyme. 2P-AMP binding was shown to trigger an unprecedented induced-fit transition in the NADP-binding domain. Prolonged incubation with NADP+ or 2P-AMP led to the formation of a disulfide-stabilized dimeric form of the enzyme, which was catalytically inactive. Insights in its three-dimensional structure indicate that PfFNR is particularly suited for the rational design of inhibitors, which could represent valuable leads in the development of antimalarial compounds.
1. Gardner, M. J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R. W., Carlton, J. M., et al. (2002) Nature 419, 498–511.
2. Foth, B. J. & McFadden, G. I. (2003) Int. Rev. Cytol. 224, 57–110.
3. Fichera, M. E. & Roos, D. S. (1997) Nature 390, 407–409.
4. Pandini, V., Caprini, G., Thomsen, N., Aliverti, A., Seeber, F. & Zanetti, G. (2002) J. Biol. Chem. 277, 48463–48471.
5. Seeber, F., Aliverti, A. & Zanetti, G. (2005) Curr. Pharm. Des. 11, 3159–3172.
6. Röhrich, R. C., Englert, N., Troschke, K., Reichenberg, A., Hintz, M., Seeber, F., Balconi, E., Aliverti, A., Zanetti, G., Kohler, U., Pfeiffer, M., Beck, E., Jomaa, H. & Wiesner, J. (2005) FEBS Lett. 579, 6433–6438
Expression of Plasmodium falciparum ferredoxin-NADP+ reductase in Escherichia coli as a cleavable fusion with the SUMO protein
No full textThe ferredoxin-NADP+ reductase of Plasmodium falciparum (PfFNR) is an enzyme similar to that found in non-photosynthetic plastids of plants (1). It is involved in the biosynthesis of isoprenoid precursors (2) and it has been proposed as a target for novel antimalarial drugs (3). In order to support in-depth inhibition studies, large amounts of recombinant PfFNR are required. To this aim, we have developed an E. coli expression system based on the fusion with the yeast SUMO protein (pET SUMO system, Invitrogen). Recombinant PfFNR has been purified by two successive steps of immobilized nickel ion affinity chromatography. Mature PfFNR has been generated during the purification procedure by treatment with recombinant SUMO protease. Although the total yield of the procedure is lowered by the tendency of the fusion product to undergo spontaneous splitting in vivo, this quick and cheap isolation protocol can provide about 30 mg highly purified PfFNR from each run.
1. Balconi, E., et al. (2009) FEBS J. 276, 3825–3836
2. Röhrich, R.C., et al. (2005) FEBS Lett. 579, 6433–6438
3. Seeber, F., et al. (2005) Curr. Pharm. Des. 11, 3159–3172
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