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Ferredoxin-NADP+ Reductase and Ferredoxin of the Protozoan Parasite Toxoplasma gondii Interact Productivelyin Vitro and in Vivo

2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês

10.1074/jbc.m209388200

1083-351X

V. Pandini, Gianluca Caprini, Nadine Thomsen, Alessandro Aliverti, Frank Seeber, Giuliana Zanetti,

Metal-Catalyzed Oxygenation Mechanisms

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP+reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH−) of the reductase was shown to be more positive than that of the NADP+/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied bothin vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondiiform a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites. Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP+reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH−) of the reductase was shown to be more positive than that of the NADP+/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied bothin vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondiiform a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites. Toxoplasma gondii, the causative agent of toxoplasmosis, is a protozoan parasite belonging to the phylum Apicomplexa (1Wong S.Y. Remington J.S. AIDS. 1993; 7: 299-316Google Scholar). This phylum comprises several other pathogens of humans or economically important animals, e.g. Plasmodium sp. (causative agent of malaria),Cryptosporidium parvum (cryptosporidiosis), andEimeria sp. Nearly all Apicomplexa possess an unique organelle called apicoplast (for reviews see Refs. 2Wilson R.J.M. J. Mol. Biol. 2002; 319: 257-274Google Scholar, 3Marechal E. Cesbron-Delauw M.F. Trends Plant Sci. 2001; 6: 200-205Google Scholar, 4Roos D.S. Crawford M.J. Donald R.G. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Google Scholar). Recent studies have established that it is related evolutionarily to plastids of photosynthetic red algae and that it was acquired by the ancestor of extant Apicomplexa by an endosymbiotic event (5Köhler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Google Scholar, 6Fast N.M. Kissinger J.C. Roos D.S. Keeling P.J. Mol. Biol. Evol. 2001; 18: 418-426Google Scholar). The circular apicoplast genomes (35 kb in size) of both T. gondii andPlasmodium falciparum have been sequenced completely (7.Toxoplasma gondii 35kb plastid DNA annotation on World Wide Web: www.ncbi.nlm.nih.gov/cgi-bin/Entrez/framik?db=genome&gi=12164.Google Scholar, 8Wilson R.J.M. Denny P.W. Preiser P.R. Roberts K. Roy A. Whyte A. Strath M. Moore D.J. Williamson D.H. J. Mol. Biol. 1997; 261: 155-172Google Scholar). Their reduced size with respect to those of plant and algal plastids suggests that loss of genes encoding apicoplast proteins or their progressive transfer to the nucleus has taken place. Proteins to be imported into the organelle possess a characteristic N-terminal bipartite targeting sequence, which is both necessary and sufficient to transport these proteins into the apicoplast (4Roos D.S. Crawford M.J. Donald R.G. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Google Scholar, 9DeRocher A. Hagen C.B. Froehlich J.E. Feagin J.E. Parsons M. J. Cell Sci. 2000; 113: 3969-3977Google Scholar, 10Waller R.F. Reed M.B. Cowman A.F. McFadden G.I. EMBO J. 2000; 19: 1794-1802Google Scholar, 11He C.Y. Striepen B. Pletcher C.H. Murray J.M. Roos D.S. J. Biol. Chem. 2001; 276: 28436-28442Google Scholar). This distinct sequence feature has recently allowed the identification of some enzymes predicted to be localized in the apicoplast (10Waller R.F. Reed M.B. Cowman A.F. McFadden G.I. EMBO J. 2000; 19: 1794-1802Google Scholar, 12Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Google Scholar, 13Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Turbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Google Scholar, 14Jelenska J. Crawford M.J. Harb O.S. Zuther E. Haselkorn R. Roos D.S. Gornicki P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2723-2728Google Scholar) and helped in the assembly of a provisory metabolic map of this organelle by whole genome analysis (15Roos D.S. Crawford M.J. Donald R.G. Fraunholz M. Harb O.S. He C.Y. Kissinger J.C. Shaw M.K. Striepen B. Philos. Trans. R. Soc. Lond.-Biol. Sci. 2002; 357: 35-46Google Scholar). Recently, both a ferredoxin-NADP+ reductase (FNR) 1The abbreviations used are: FNR, ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2); TgFNR, T. gondii FNR; 6H-L/TgFNR, His-tagged longer form of TgFNR; S/TgFNR, shorter form of TgFNR; SoFNR, S. oleracea leaf FNR; ZmFNR, Z. mays root FNR; Fd, ferredoxin; TgFd, T. gondii Fd; PfFd, P. falciparum Fd; SoFdI, S. oleracea Fd I; Fdox, oxidized Fd; Fdred, reduced Fd; NLS, nuclear localization signal; Ni-NTA, nickel-nitrilotriacetic acid; FPLC, fast protein liquid chromatography. of T. gondii and a [2Fe-2S] ferredoxin (Fd) of P. falciparum have been cloned (16Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Google Scholar). They both contain a putative N-terminal apicoplast targeting signal that was shown in the case ofT. gondii FNR (TgFNR) to be sufficient to target a reporter protein into the apicoplast (17Striepen B. Crawford M.J. Shaw M.K. Tilney L.G. Seeber F. Roos D.S. J. Cell Biol. 2000; 151: 1423-1434Google Scholar). TgFNR starting from residue 150 of the whole coding sequence was expressed in Escherichia coli, and the enzyme was shown to be active as an NADPH-dependent oxidoreductase (16Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Google Scholar). Phylogenetic analysis indicated that TgFNR is more similar to the isoforms present in non-photosynthetic plastids than to those of cyanobacteria or chloroplasts. In the latter cases, FNR is responsible for the electron transfer from photosystem I to NADP+, according to Equation1. 2Fdred+NADP++H+↔2Fdox+NADPHEquation 1 NADPH is used subsequently in the Calvin cycle (18Arakaki A.K. Ceccarelli E.A. Carrillo N. FASEB J. 1997; 11: 133-140Google Scholar). In non-photosynthetic plastids electrons flow in the reverse direction, from NADPH to Fdox yielding Fdred (19Bowsher C.G. Hucklesby D.P. Emes M.J. Plant J. 1993; 3: 463-467Google Scholar), which then serves as a reductant for various enzymes, i.e. nitrite reductase, glutamate synthase, sulfite reductase, and lipid desaturases (20Neuhaus H.E. Emes M.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 111-140Google Scholar, 21Knaff D.B. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, The Netherlands1996: 333-361Google Scholar). By analogy, the redox system NADPH/FNR/Fd of the apicoplast would be predicted to provide reducing power to biosynthetic pathways yet to be identified. It is assumed that the apicoplast has retained a number of parasite-specific metabolic tasks, and because the vertebrate hosts of Apicomplexa do not possess a homolog of this organelle, these metabolic pathways are therefore prime targets for efficient and specific anti-infection drugs (22McFadden G.I. Roos D.S. Trends Microbiol. 1999; 7: 328-333Google Scholar). In this respect the FNR/Fd redox system is a promising candidate, because it is presumably present in the apicoplast of most, if not all, apicomplexan parasites (23Seeber F. Int. J. Parasitol. 2002; 32: 1207-1217Google Scholar). A detailed knowledge of the biochemical properties of apicomplexan FNR and its specific interaction with Fd is a prerequisite for the development of inhibitory substances of this redox couple. Here we report on the overproduction and purification of TgFNR, as well as T. gondii Fd (TgFd). Their subsequent biochemical characterization allowed us to highlight several distinctive differences of the parasite FNR/Fd redox couple compared with their plant counterparts, especially with regard to the FAD cofactor redox potential, the catalytic efficiency, and the protein-protein interactions. All chemicals and pyridine nucleotides were purchased from Sigma-Aldrich. All chromatographic columns and media were purchased from Amersham Biosciences, with the exception of Ni-NTA-agarose, which was from Qiagen. Cytochrome c (C2506; Sigma) was purified further by ion-exchange chromatography on SP-Sepharose. Restriction endonucleases, DNA polymerase, and DNA modifying enzymes were supplied by Amersham Biosciences or New England Biolabs. Recombinant spinach (Spinacia oleracea) ferredoxin I (SoFdI), spinach FNR (SoFNR), and maize (Zea mays) root FNR (ZmFNR) were purified as reported previously (24Piubelli L. Aliverti A. Bellintani F. Zanetti G. Protein Expr. Purif. 1995; 6: 298-304Google Scholar, 25Aliverti A. Corrado M.E. Zanetti G. FEBS Lett. 1994; 343: 247-250Google Scholar, 26Aliverti A. Faber R. Finnerty C.M. Ferioli C. Pandini V. Negri A. Karplus P.A. Zanetti G. Biochemistry. 2001; 40: 14501-14508Google Scholar). For expression of TgFNR and TgFd, their mature coding sequence was placed under the control of the T5-based, isopropyl-1-thio-β-d-galactopyranoside-inducible hybrid promoter PN25/03/04 in vector pDS56/RBSII (27Bujard H. Gentz R. Lanzer M. Stueber D. Mueller M. Ibrahimi I. Haeuptle M.T. Dobberstein B. Methods Enzymol. 1987; 155: 416-433Google Scholar), yielding plasmids pS1-S/TgFNR and pS1-TgFd, respectively (see Fig. 1). A commercial version of the above vector, pQE31 (Qiagen) served as recipient for a longer version of the coding sequence of TgFNR starting at Leu-121 (yielding pQE-L/TgFNR; see Fig. 1). The coding sequence of TgFd was taken from expressed sequence tag clone BG659097 (which is derived from mRNA of the VEG strain), and its insert was sequenced further and shown to encode all of the mature TgFd and to possess a long N-terminal extension but without an initiator AUG. A lexA-based yeast two-hybrid system was used for the detection of protein-protein interactions in vivo (28Fashena S.J. Serebriiskii I.G. Golemis E.A. Methods Enzymol. 2000; 328: 14-26Google Scholar). The shorter TgFNR was cloned as an EcoRI fragment into pB42AD (Origene), and the different ferredoxins were cloned into the EcoRI site of pGILDA (Clontech). Plasmids pTUK-PfFd (16Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Google Scholar), pETFdI (24Piubelli L. Aliverti A. Bellintani F. Zanetti G. Protein Expr. Purif. 1995; 6: 298-304Google Scholar), and pS1-TgFd (see above) served as source for the ferredoxins. In both two-hybrid plasmids expression is driven by a galactose-dependent promoter (see Fig. 1). All constructs were verified by DNA sequencing in the newly assembled parts of the vector. Sequence alignments were performed using ClustalX and optimized by visual inspection (29Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Google Scholar). E. coliRRIΔM15 cells transformed with expression plasmids (pTUK-TgFNR, pS1-S/TgFNR, pQE-L/TgFNR, pS1-TgFd) were grown in flasks under vigorous shaking in 2× YT medium supplemented with 100 mg/liter ampicillin. For protein purification, E. coli cells were grown at 30 °C (for TgFNRs) or 37 °C (for TgFd) in a New Brunswick 12-liter fermentor to midlog phase (A600 = 1.2–1.5). Then the culture was induced with 0.1 mm isopropyl β-d-thiogalactopyranoside, and cells were harvested after 4 h (for TgFNR) or 2 h (for TgFd) of further growth. All purification steps were performed at 4 °C except for FPLC, which was carried out at room temperature. E. coli cell paste was resuspended in 2.5 ml/g of Buffer A (50 mm sodium phosphate, pH 8.0, containing 300 mm NaCl, 10 mm imidazole, and 1 mm 2-mercaptoethanol supplemented with 1 mmphenylmethylsulfonyl fluoride and disrupted by sonication. After removal of cell debris by centrifugation at 43,000 × gfor 1 h, the soluble fraction was loaded on an Ni-NTA-agarose column pre-equilibrated with Buffer A. The molarity of imidazole in Buffer A was brought to 20 mm and then used for extensive washing. Elution of recombinant proteins was then performed with 250 mm imidazole in Buffer A. In the case of TgFNR, the pooled enzyme-containing fractions were supplemented with 5 mmEDTA and dialyzed against 25 mm sodium phosphate, pH 7.0, containing 10% glycerol, 1 mm EDTA, and 1 mm2-mercaptoethanol. TgFNR was then loaded on a HiLoad SP-Sepharose high performance column and eluted with 10 column volumes of a linear gradient from 0 to 1 m NaCl. The purified TgFNR was stored at −20 °C in 150 mm Tris-HCl, pH 7.0, containing 10% glycerol, 0.5 mm EDTA, and 1 mm dithiothreitol. In the case of TgFd, the protein eluted from the Ni-NTA-agarose column was concentrated by ultrafiltration and gel-filtered on a Superdex 75 column equilibrated in 150 mm Tris-HCl, pH 8.0, containing 1 m NaCl and 10% glycerol. The purified TgFd was stored at −80 °C under nitrogen in 150 mm Tris-HCl, pH 7.0, containing 10% glycerol. SDS-PAGE was carried out on 12% polyacrylamide gels. Analytical gel-filtration analyses were performed on an ÄKTA FPLC apparatus equipped with a Superose 12 column in 25 mm sodium phosphate, pH 7.0, containing 10% glycerol, 0.5 mm EDTA, and 2 mm2-mercaptoethanol. Absorption spectra were recorded with an Agilent 8453 diode-array spectrophotometer. The extinction coefficient of the protein-bound flavin was determined spectrophotometrically by quantitating the FAD released from the apoprotein following SDS treatment (30Aliverti A. Curti B. Vanoni M.A. Methods Mol. Biol. 1999; 131: 9-23Google Scholar). Fluorescence measurements were performed on a Jasco FP-777 spectrofluorometer at 15 °C. The identity of the enzyme-bound flavin was assessed fluorimetrically by treating with phosphodiesterase the flavin released after thermal denaturation at 100 °C of the TgFNR (30Aliverti A. Curti B. Vanoni M.A. Methods Mol. Biol. 1999; 131: 9-23Google Scholar). Enzyme-catalyzed reactions were monitored continuously on an Agilent 8453 diode-array spectrophotometer. Standard diaphorase activity was measured in 0.1 m Tris-HCl, pH 8.2, at 25 °C with K3Fe(CN)6 as electron acceptor and NADPH or NADH as reductants. Standard cytochrome creductase activity was assayed in the same buffer as above with either 7 or 9 μm SoFdI or TgFd, using 40 μmpurified cytochrome c as the terminal electron acceptor. The NADPH concentration was kept constant by regeneration with 2.5 mm glucose 6-phosphate and 2 μg/ml glucose-6-phosphate dehydrogenase. Steady-state kinetic parameters for the Fd-dependent cytochrome c reductase activity were determined by varying the concentrations of both NADPH and Fd. Initial rate data (v) were fitted by non-linear regression using Grafit 4.0 software (Erythacus Software Ltd., Staines, United Kingdom) to a Michaelis-Menten equation. All reduction experiments were carried out in an anaerobic cuvette at 15 °C. Solutions were made anaerobic by successive cycles of equilibration with O2-free nitrogen and evacuation. Photoreduction of TgFNR using EDTA/light (31Massey V. Hemmerich P. J. Biol. Chem. 1977; 252: 5612-5614Google Scholar) were performed in 50 mm sodium phosphate, pH 7.0, containing 15 mm EDTA and 1.5 μm 5-carba-5-deazariboflavin. To monitor the in vivo interactions between TgFNR and various Fds the protocol of Fashena et al. (28Fashena S.J. Serebriiskii I.G. Golemis E.A. Methods Enzymol. 2000; 328: 14-26Google Scholar) was followed. Yeast strain EGY48 (MATα trp1 his3 ura3 leu2::6 LexAop-LEU2; Origene) containing the lacZ reporter plasmid pSH18-34 was used as recipient for all plasmids. As negative, non-interactive proteins two control plasmids from the supplier (Origene) were used. Interaction-dependent growth was assessed by plating the transformed cells onto His/Trp/Leu drop-out medium containing either 2% glucose or 2% galactose. Liquid β-galactosidase assays were performed in 96-well plates using Y-PER yeast lysis solution (Pierce) ando-nitrophenyl-β-d-galactopyranoside (Sigma) as enzyme substrate, following the instructions of the manufacturer (Pierce). Protein cross-linking was performed as described previously (32Zanetti G. Morelli D. Ronchi S. Negri A. Aliverti A. Curti B. Biochemistry. 1988; 27: 3753-3759Google Scholar). FNR and Fd (8 and 40 μm, respectively) were cross-linked by treatment with 5 mm N-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 25 mm sodium phosphate, pH 7.0. Affinity chromatography of FNRs was performed on Fd-Sepharose 4B. Immobilized SoFdI was obtained by coupling 250 nmol of SoFdI to 350 mg of CNBr-activated-Sepharose 4B following the manufacturer's directions. SoFdI-conjugated resin (1 ml) was packed and used as an affinity column in an FPLC apparatus. FNRs (∼10 nmol) were loaded on the affinity column, equilibrated in 20 mm HEPES, pH 7.5, containing 10% glycerol and 1 mm dithiothreitol. After washing with the same buffer, FNRs were eluted with a linear gradient of NaCl. A second type of experiment was performed under the same conditions except for the presence of 2 m urea in all the buffers and samples. Several constructs were designed and utilized for the production of TgFNR inE. coli. The originally described plasmid pTUK-TgFNR, which allows expression of TgFNR as a fusion protein with His6-tagged yeast ubiquitin, starting at Ser-150 of the cDNA-derived FNR sequence (16Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Google Scholar), was found unsuitable for high level purification. A second plasmid was constructed in which the above coding sequence contained only an N-terminal His6 tag for purification (Fig. 1). Although for both plasmids the recombinant TgFNR was found soluble and active, the purified enzymes thus obtained were nevertheless prone to precipitation and underwent proteolysis during storage. Furthermore, a small percentage (∼4%) of the enzyme contained a modified flavin cofactor, which was identified as 6-OH-FAD (33Pandini V. Caprini G. Aliverti A. Zanetti G. Chapman S. Perham R. Scrutton N. Flavins and Flavoproteins. Rudolf Weber, Agency for Scientific Publications, Berlin2002: 899-904Google Scholar). With the aim of stabilizing the protein, Leu-121 of TgFNR, which encompasses a region well conserved in non-photosynthetic FNRs (16Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Google Scholar), was chosen as the starting point for the longer mature form (Fig. 1). Again an N-terminal His6 tag was added to facilitate purification of the protein, resulting in plasmid pQE-L/TgFNR. Notwithstanding the low expression level obtained for this protein, a two-step procedure (affinity chromatography on Ni-NTA-agarose followed by ionic-exchange chromatography on SP-Sepharose) led to the production of 2 mg of purified enzyme/liter of culture (named 6H-L/TgFNR). 6H-L/TgFNR migrates in SDS-PAGE as a band of ∼43 kDa (see Fig. 6, lane 5). The molecular mass value of the native enzyme as analyzed by FPLC on a Superdex 75 column was 43 kDa ± 2, well in keeping with that calculated from the deduced amino acid sequence (43.4 kDa, including the His6 tag). The gel filtration conditions were chosen to avoid possible aggregation phenomena, i.e. 10% glycerol and 1 mm dithiothreitol were added to the buffer. In fact, there are eight cysteines present in the deduced sequence of TgFNR. By treatment of the enzyme with 5,5′-dithiobis(2-nitrobenzoate) under denaturing conditions, all the cysteines were titrated, indicating that no disulfide bonds are present, whereas modification of 6H-L/TgFNR under non-denaturing conditions yielded only three readily titratable cysteines. This suggests that these latter residues are exposed to solvent and hence they could possibly form intermolecular disulfide bonds by oxidation, resulting in protein aggregation. The absorption spectrum of the purified protein is that typical of a flavoprotein, with bands centered at 395 and 454 nm (33Pandini V. Caprini G. Aliverti A. Zanetti G. Chapman S. Perham R. Scrutton N. Flavins and Flavoproteins. Rudolf Weber, Agency for Scientific Publications, Berlin2002: 899-904Google Scholar). Maximal absorbance in the UV region was at 272 nm. A value of 6.4 for theA272/A454 ratio was calculated from the spectrum. Flavin fluorescence was quenched almost completely. The non-covalently-bound flavin in 6H-L/TgFNR was shown to be FAD. The extinction coefficient of the enzyme at 454 nm was calculated to be 10.1 mm−1 cm−1from the amount of FAD released after protein denaturation by SDS. We explored the anaerobic reduction of the flavin cofactor bound to the enzyme to obtain information about its redox potential and the intermediate species formed during reduction. Stepwise anaerobic photoreductions of 6H-L/TgFNR were performed for the free enzyme (Fig. 2A) and in the presence of an equimolar amount of NADP+ (Fig. 2B). During reduction, the neutral blue semiquinone accumulated, as is the case for the plant FNRs. When NADP+ was present in the anaerobic cuvette, long wavelength bands absorbing well beyond 700 nm, which are ascribed to charge transfer species between NADP(H) and FAD(H−), were formed. Data obtained in this latter type of experiments were used to calculate the concentrations of NADPH and fully reduced flavin at several photoreduction times. These results are plotted in Fig. 3, and data obtained in the same type of experiments for the spinach leaf and the maize root enzymes are added for comparison. The course of reduction in the case of 6H-L/TgFNR highlighted that nearly full reduction of the FAD cofactor ensued before NADPH started to accumulate significantly. This indicates that the value of the enzyme redox potential is more positive than that of the NADP+/NADPH couple. Furthermore, comparison with the data obtained in the case of the two plant FNRs shows that 6H-L/TgFNR has a quite distinctive behavior; its pattern was nearly exactly specular with respect to that of the photosynthetic FNR, whereas the non-photosynthetic plant FNR showed an intermediate behavior. Shifting of the curve from left to right in this type of plot indicates that the redox potential of the enzyme FAD cofactor becomes less negative going from the photosynthetic to the non-photosynthetic isoform and to TgFNR. This is in keeping with the different physiological roles of the different isoforms.Figure 3Photoreduction of different FNR forms in the presence of NADP+: relationship between NADP+and FAD reduction. Absorbance at 340 nm, which is practically an isosbestic point for the various reduction forms of FNR, is used to monitor NADPH formation. At each time of irradiation, the ΔA340, as a fraction of the maximal ΔA340, was plotted against the ratio of fully reduced enzyme FAD to the sum of fully reduced and oxidized enzyme flavin. The data were corrected for the presence of the FAD semiquinone. ●, SoFNR; ■, ZmFNR; ○, 6H-L/TgFNR.View Large Image Figure ViewerDownload (PPT) The physiological activity of the plant homologs of TgFNR is to catalyze electron transfer between NADP(H) and a [2Fe-2S] ferredoxin. To test for such activity we initially used FdI of spinach chloroplasts (photosynthetic isoform, SoFdI) because of difficulties in the production of substantial amounts of recombinant P. falciparum Fd described earlier (16Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Google Scholar). More recently, however, we could identify a single expressed sequence tag clone in the T. gondiiexpressed sequence tag database that encoded the whole mature TgFd sequence, evidenced by its high sequence identity to other plant-type Fds (Fig. 4, A andB) and part of the N-terminal apicoplast targeting domain (data not shown). The latest release of ToxoDB 2.0 (ToxoDB.org) contains a genomic contig (TGG_640), which encompasses the wholeT. gondii Fd sequence including its complete N terminus with a predicted signal peptide (data not shown). The putative mature form of TgFd was expressed as an His6 tag fusion protein (see Fig. 1). Analysis of E. coli cell extracts revealed that only part of the synthesized TgFd was recovered as the holoprotein in the soluble fraction. The purification procedure required a second step after the Ni-NTA affinity chromatography. Gel filtration on Superdex 75 yielded a homogeneous holoprotein as judged by native PAGE and with a molecular mass of ∼20 kDa by SDS-PAGE (see Fig. 6, lane 5). The procedure led to the production of 1 mg of purified protein/liter of culture. TgFd showed the typical spectrum of a [2Fe-2S] ferredoxin with peaks at 277, 333, 424, and 464 nm (data not shown). A value of 0.60 for theA424/A277 ratio was determined. As expected, 6H-L/TgFNR was very active as an NADPH-dependent diaphorase. Kinetic parameters for the ferricyanide reductase reaction were determined; values for kcat andKmNADPH were 700 ± 5 e− eq/s and 34 μm ± 1, respectively. 6H-L/TgFNR differentiates strongly between NADPH and NADH, showing very low ferricyanide reduction activity even with 2 mm NADH. An approximate value for NADPH preference over NADH was 180,000-fold. The kinetic parameters for the NADPH-cytochrome creductase activity of 6H-L/TgFNR with both SoFdI and TgFd are compared in Table I. Clearly, there is a preference of 6H-L/TgFNR for the parasite iron-sulfur protein. We also determined the kinetic parameters of plant FNR isoforms using TgFd as the protein substrate to evaluate the specificity of the protein-protein interactions in these various redox couples (Table II). Apparently, 6H-L/TgFNR showed a kcat value half of that of the plant enzymes when TgFd was used as electron acceptor and even lower when SoFdI was used. On the other hand, the Kmvalue of 6H-L/TgFNR for TgFd was ∼3-fold lower than that for leaf SoFdI (Table I). Thus, the catalytic efficiency of the T. gondii couple was twice that of the leaf homolog (32 e− eq/s μm; see Ref. 34Piubelli L. Aliverti A. Bellintani F. Zanetti G. Eur J Biochem. 1996; 236: 465-469Google Scholar) or of the heterologous couples (Table II). SoFNR showed the same efficiency with both ferredoxins in this type of reaction.Table IKinetic parameters for the Fd-dependent NADPH-cytochrome c reductase reaction catalysed by 6H-L/TgFNRFd formkcatK Fdmkcat/Kme− eq s−1μme− eq s−1μm−1SoFdI32 ± 15 ± 0.56.4 ± 0.8TgFd115 ± 51.9 ± 0.261 ± 9 Open table in a new tab Table IIKinetic parameters for the TgFd-dependent NADPH-cytochrome c reductase reaction catalysed by different FNR formsFNR formkcatK TgFdmkcat/Kme− eq s−1μme− eq s−1μm−1SoFNR180 ± 155 ± 0.336 ± 5ZmFNR227 ± 67 ± 0.432 ± 3 Open table in a new tab Because electron transfer to Fd relies absolutely on the physical interaction between Fd and FNR, we wished to investigate this aspect in more detail. To date all protein-protein interaction studies between Fds and FNRs in any system have only been performed in vitro. To extend the experimental possibilities to the in vivo situation we established a yeast two-hybrid system consisting of S/TgFNR (note that this is the shorter version; see Fig. 1) and either SoFdI or the two apicomplexan Fds (Fig. 1). In the used two-hybrid format both genes are under control of a galactose-inducible promoter so that a potential interaction can only be observed under inducing conditions. Using this system and leucine auxothrophy as marker for a positive protein-protein interaction we observed that S/TgFNR can interact with all three Fds invivo, although seemingly to a different extent as judged by the difference in cell mass on selective medium (Fig. 5A). According to this experiment, SoFdI bound better to S/TgFNR than did TgFd, and PfFd reproducibly interacted only weekly. Using a second reporter as a measurement for interaction strength we determined the β-galactosidase activity of individual yeast clones after cross-mating them to a strain containing a lexA-driven lacZ reporter plasmid. Again SoFdI appeared to be more efficient in interacting with S/TgFNR than did TgFd or PfFd (Fig. 5B). This was