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 kcat, 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 kcat. 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 mNADPH and Kd 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.
Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis / D. Crobu, G. Canevari, M. Milani, V. Pandini, M.A. Vanoni, M. Bolognesi, G. Zanetti, A. Aliverti. - In: BIOCHEMISTRY. - ISSN 0006-2960. - 48:40(2009 Oct 13), pp. 9525-9533.
|Titolo:||Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis|
CROBU, DANILA (Primo)
ZANETTI, GIULIANA (Penultimo)
ALIVERTI, ALESSANDRO (Corresponding)
|Parole Chiave:||binding sites; catalysis; crystallography, x-ray; ferredoxin-nadp reductase; histidine; kinetics; lysine; nadp; niacinamide; plasmodium falciparum; protein binding; protein subunits; substrate specificity; biochemistry|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore BIO/11 - Biologia Molecolare
|Data di pubblicazione:||13-ott-2009|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1021/bi9013209|
|Appare nelle tipologie:||01 - Articolo su periodico|