| 2000 |
FNR (plant-type ferredoxin-NADP+ reductase) from Apicomplexan parasites is nuclear-encoded but localizes to the apicoplast, bearing an N-terminal bipartite transit peptide domain. The recombinant protein was obtained in active form, and the system is proposed to supply reduced ferredoxin for biosynthetic processes in the apicoplast. |
Cloning, recombinant protein expression, Western blot showing unprocessed and native forms indicative of apicoplast import/processing, phylogenetic analysis |
The Journal of biological chemistry |
Medium |
11056177
|
| 2005 |
FNR and ferredoxin of P. falciparum constitute an NADPH-dependent electron transfer chain that serves as the physiological electron donor for LytB (the last enzyme of the DOXP/MEP isoprenoid biosynthesis pathway). LytB and ferredoxin were shown to form a stable protein complex, and LytB was catalytically active only in the presence of the NADPH→FNR→Fd electron transfer system. |
In vitro reconstitution of electron transfer pathway, stable complex formation demonstrated between LytB and ferredoxin, enzymatic activity assay |
FEBS letters |
High |
16289098
|
| 2007 |
Recombinant P. falciparum FNR transfers electrons selectively to P. falciparum ferredoxin in a reconstituted NADPH-dependent cytochrome c reduction system, demonstrating that an NADPH→FNR→Fd cascade is operative. The crystal structure of P. falciparum ferredoxin (its redox partner) was solved, showing backbone similarity to plant ferredoxins but with unique surface charge distribution. |
Recombinant protein production (synthetic genes), NADPH-dependent cytochrome c reduction assay, X-ray crystallography of Fd |
Journal of biochemistry |
High |
17251200
|
| 2007 |
Crystal structures of P. falciparum FNR were solved in free form (2.4 Å) and in complex with 2'-phospho-AMP (2.7 Å). The structures reveal a covalent dimer stabilized by an intermolecular disulfide bond at Cys99. Binding of 2'-phospho-AMP (mimicking the adenylate moiety of NADP(H)) triggers a helix-coil transition in the NADP-binding domain. NADP+ and 2'-phospho-AMP promote dimer formation in solution, and the isolated dimer is essentially inactive; full activity is recovered upon disulfide reduction. |
X-ray crystallography, kinetic characterization, ligand binding studies, solution studies with oxidizing/reducing agents |
Journal of molecular biology |
High |
17258767
|
| 2007 |
NMR chemical shift perturbation mapping and site-directed mutagenesis of ferredoxin identified two acidic regions on P. falciparum Fd (Asp26/Glu29/Glu34 and Asp65/Glu66) as the dominant electrostatic interaction sites with FNR. Glu29 is the single most important residue; mutations in these regions reduced electron transfer activity and affinity for FNR. |
NMR chemical shift perturbation, site-directed mutagenesis of Fd, electron transfer activity assay, affinity chromatography |
Journal of biochemistry |
High |
17938142
|
| 2009 |
P. falciparum FNR displays a kcat five-fold lower than typical plastidic FNRs. Rapid kinetics showed that hydride transfer between NADPH and FAD is slower than in homologues, and the FAD semiquinone species is highly destabilized. These two features account for the enzyme's poor catalytic efficiency. Under oxidizing conditions (H2O2), FNR forms an inactive disulfide-stabilized dimer; glutathione and lipoate can reverse this, suggesting redox-regulated quaternary structure as a possible regulatory mechanism in the apicoplast. |
Steady-state and rapid-reaction kinetics, redox potential determination, oxidizing/reducing agent treatments |
The FEBS journal |
High |
19523113
|
| 2009 |
P. falciparum FNR catalyzes single-electron reduction of quinones (redox cycling), contributing to antiplasmodial activity. The presence of aziridinyl groups on quinones does not enhance redox cycling by FNR; instead aziridinyl-quinones likely act as DNA-alkylating agents after two-electron reduction, which can be carried out by single-electron flavoenzymes including FNR under partial anaerobiosis. |
In vitro single-electron reduction rate measurements, multiparameter statistical analysis of antiplasmodial activity |
Archives of biochemistry and biophysics |
Medium |
19919822
|
| 2009 |
His286 in the NADP-binding site of P. falciparum FNR plays a dual role: it provides binding energy for NADPH and favors the catalytically competent orientation of the nicotinamide ring for hydride transfer. Replacement with aliphatic residues decreased both NADPH affinity and kcat; replacement with Gln produced a more active enzyme; replacement with Lys destabilized the nicotinamide-isoalloxazine interaction. Lys249 participates in NADPH recognition via interaction with the 2'-phosphate group. |
Site-directed mutagenesis, kinetic characterization, crystal structures of mutant-2'-P-AMP complexes |
Biochemistry |
High |
19736991
|
| 2012 |
The conserved Tyr258 hydroxyl group of P. falciparum FNR, which interacts with the 2'-phosphate of NADPH, is the primary determinant of coenzyme specificity. The Y258F mutation selectively decreased kcat for NADPH-dependent reactions ~2-fold (matching the NADH rate), increased Km for NADPH 4-fold, decreased Km for NADH 3-fold, and reduced coenzyme discrimination from 70-fold to 1.5-fold. Rapid kinetics and active-site titrations indicated the effect arises from destabilization of the catalytically competent conformation of bound NADPH. |
Site-directed mutagenesis, steady-state kinetics, rapid-reaction kinetics, active-site titrations, anaerobic photoreduction |
Biochemistry |
High |
22519987
|
| 2018 |
Basic amino acid residues on the FAD-binding face of P. falciparum FNR, including residues within a Plasmodium-specific insertion region, are important for interaction with P. falciparum ferredoxin. Comprehensive substitution to acidic residues showed that these basic residues are Plasmodium-specific (not conserved in plants/cyanobacteria), explaining the higher affinity and specificity of PfFd:PfFNR interaction compared to plant systems. |
Site-directed mutagenesis of FNR, kinetic analysis of electron transfer, affinity chromatography to immobilized Fd, thermodynamic binding measurements (ITC or equivalent) |
Journal of biochemistry |
High |
29688515
|
| 2020 |
P. falciparum FNR catalyzes single-electron reduction of quinones, nitroaromatic compounds, and aromatic N-oxides via a ping-pong mechanism. Reactivity of all compound classes increases with their single-electron reduction midpoint potential (E17). For quinones, oxidation of FADH• to FAD is the possible rate-limiting step during FAD reoxidation. Ferredoxin stimulates the quinone- and nitro-reductase reactions of FNR by providing an additional reduction pathway via reduced Fd. |
In vitro kinetic study of single-electron reduction, ping-pong mechanism analysis, electron transfer distance calculations |
International journal of molecular sciences |
Medium |
32370303
|
| 2021 |
P. falciparum FNR catalyzes redox cycling of the antimalarial compound plasmodione (PD) in the presence of NADPH and oxygen, generating both key metabolites PD-bzol (benzylic alcohol) and PDO (3-benzoylmenadione) via a semiquinone radical intermediate. This FNR-mediated redox cycling generates reactive oxygen species (ROS) in the apicoplast, as demonstrated using a genetically encoded glutathione redox sensor in live parasites. |
In vitro enzyme redox cycling assay, LC-MS metabolite detection, live parasite imaging with genetically encoded redox sensor (hGrx1-roGFP2) in transgenic 3D7 P. falciparum |
ACS infectious diseases |
High |
33855850
|
| 2021 |
Three chemically reactive residues are important for P. falciparum FNR activity: Cys99 (active-site, responsible for hydride transfer), Cys284 (whose oxidation leads to inactive dimer), and His286 (NADPH binding). Both cysteines are promising targets for covalent inhibition, as shown using residue-specific chemical reagents. |
Site-directed mutagenesis, residue-specific chemical modification, enzymatic activity assays |
Biochemical and biophysical research communications |
Medium |
34509083
|
| 2022 |
Genetic deletion of FNR in P. falciparum (using an apicoplast metabolic bypass line) demonstrated that FNR is essential for blood-stage parasite survival but is not required for apicoplast maintenance. The Fd/FNR system provides reducing power to certain downstream iron-sulfur cluster proteins that are individually essential for parasite survival. |
Genetic deletion (conditional KO in PfMev apicoplast bypass line), growth/viability assays, double deletion epistasis with FeS transfer proteins SufA and NfuApi |
mBio |
High |
35164549
|
| 2025 |
P. falciparum FNR (ptFNR) can functionally replace the endogenous E. coli flavodoxin reductase in an E. coli complementation system, where bacterial growth depends on ptFd, ptFNR, and IspH from P. falciparum reconstituting the last step of the MEP isoprenoid biosynthesis pathway. This validated an in situ screening platform for MEP pathway inhibitors and functional mutations. |
E. coli complementation (genetic reconstitution), bacterial growth assays, LC-MS metabolite analysis, validation in conditional T. gondii Fd KO strain |
The Journal of biological chemistry |
High |
40967433
|