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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
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
Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme
No full textTwo isoforms of ferredoxin-NADP(+) reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure-function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP(+) with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k(cat)/K(m)), as an NADPH-dependent diaphorase, higher than those of both the root ( approximately 2-fold) and the leaf enzyme ( approximately 5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR
Interaction of the soluble recombinant PsaD subunit of spinach photosystem I with ferredoxin I
No full textPhotosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94
Analysis of the major species-specific insertion of Toxoplasma gondii ferredoxin-NADP(+) reductase by protein engineering
No full textRenalase : a new human flavoprotein possibly involved in the regulation of cardiac function and blood pressure
No full textRenalase, a putative monoamine oxidase, has been recently identified by the group of G.V. Desir (1). It is considered to be a new renal hormone which is involved in the regulation of cardiac function and blood pressure by way of its assumed monoamino oxidase activity. The renalase protein contains a signal peptide for secretion (1-17 aa) overlapping a FAD binding region (3-42 aa) and an amino oxidase region (75-335 aa). The similarity to MAO A and B is only 13% and it is restricted to the first 80-100 aa. We have expressed the full-length human cDNA in Escherichia coli and purified the soluble protein. Here we report on its biochemical properties. We have demonstrated for the first time that indeed this renalase is a flavoprotein and shown that contains non-covalently bound FAD at variance with the MAO enzymes. Furthermore, our protein is devoid of catecholamine oxidase activity, thus casting doubts on the real substrate of this enzyme. 1. Xu J, Li G, Wang P, Velazquez H, Yao X, Li Y, Wu Y, Peixoto A, Crowley S, Desir GV., J Clin Invest (2005), 115(5):1275-80
The elusive role played by the side chain of Lys249 of Plasmodium falciparum ferredoxin-NADP+ reductase in NADPH/NADH selectivity
No full textPlasmodium falciparum, the causative agent of tropical malaria, possesses plant-type ferredoxin-NADP+ reductase (PfFNR) and ferredoxin (PfFd), which have been proposed as targets for novel antimalarial drugs (1). The catalytic mechanism of PfFNR is under study in order to provide the bases for the design of effective enzyme inhibitors (2). Although PfFNR is quite specific for NADPH, no structural evidence was obtained for protein positive groups interacting with the 2’-phosphate of this substrate (3). We have found that change of Lys249 to Ala decreased the kcat/Km for NADPH by a factor of 10, without affecting the activity of the enzyme with NADH. In addition, PfFNR-K249A displayed an affinity for NADP+ 8-fold lower than those of the wild-type enzyme. We conclude that the side-chain of Lys249 actually interacts with the 2’-phosphate of NADPH during the catalytic cycle, and that it is part of a region flexible enough to adopt in the crystal form of the protein a conformation where such interaction is severed 1. Seeber, F., et al. (2005) Curr. Pharm. Des. 11, 3159-3172.
2. Crobu, D., et al. (2009) Biochemistry 48, 9525-9533.
3. Milani, M., et al. (2007) J. Mol. Biol. 367, 501-513
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