title: Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis.
abstract: The NADP-binding site of Plasmodium falciparum ferredoxin-NADP(+) reductase contains two basic residues, His286 and Lys249, conserved within the Plasmodium genus, but not in other plant-type homologues. Previous crystal studies indicated that His286 interacts with the adenine ring and with the 5'-phosphate of 2'-P-AMP, a ligand that mimics the adenylate moiety of NADP(H). Here we show that replacement of His286 with aliphatic residues results both in a decrease in the affinity of the enzyme for NADPH and in a decrease in k(cat), due to a lowered hydride-transfer rate. Unexpectedly, the mutation to Gln produces an enzyme more active than the wild-type one, whereas the change to Lys destabilizes the nicotinamide-isoalloxazine interaction, decreasing k(cat). On the basis of the crystal structure of selected mutants complexed with 2'-P-AMP, we conclude that the His286 side chain plays a dual role in catalysis both by providing binding energy for NADPH and by favoring the catalytically competent orientation of its nicotinamide ring. For the latter function, the H-bonding potential rather than the positively charged state of the His286 imidazole seems sufficient. Furthermore, we show that the Lys249Ala mutation decreases K(m)(NADPH) and K(d) for NADP(+) or 2'-P-AMP by a factor of 10. We propose that the Lys249 side chain participates in substrate recognition by interacting with the 2'-phosphate of NADP(H) and that this interaction was not observed in the crystal form of the enzyme-2'-P-AMP complex due to a conformational perturbation of the substrate-binding loop induced by dimerization.
authors.affiliation: Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.
authors.forename: Danila
authors.initials: D
authors.lastname: Crobu
authors.forename: Giulia
authors.initials: G
authors.lastname: Canevari
authors.forename: Mario
authors.initials: M
authors.lastname: Milani
authors.forename: Vittorio
authors.initials: V
authors.lastname: Pandini
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: Vanoni
authors.forename: Martino
authors.initials: M
authors.lastname: Bolognesi
authors.forename: Giuliana
authors.initials: G
authors.lastname: Zanetti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Aliverti
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D009536:Niacinamide / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D021122:Protein Subunits; D009536:Niacinamide; D006639:Histidine; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D008239:Lysine