3JQR

Crystal structure of the H286L mutant of Ferredoxin-NADP+ reductase from Plasmodium falciparum

Summary for 3JQR

• Entry DOI: 10.2210/pdb3jqr/pdb
• Related: 3JQP 3JQQ
• Descriptor: Ferredoxin NADP reductase, FLAVIN-ADENINE DINUCLEOTIDE (3 entities in total)
• Functional Keywords: ferredoxin-nadp+ reductase, fad, oxidoreductase
• Biological source: Plasmodium falciparum
• Total number of polymer chains: 1
• Total formula weight: 38076.55

Authors

Canevari, G.,Milani, M.,Bolognesi, M. (deposition date: 2009-09-07, release date: 2010-01-12, Last modification date: 2024-10-16)

Primary citation

Crobu, D.,Canevari, G.,Milani, M.,Pandini, V.,Vanoni, M.A.,Bolognesi, M.,Zanetti, G.,Aliverti, A.
Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis
Biochemistry, 48:9525-9533, 2009

PubMed 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.

PubMed: 19736991
DOI: 10.1021/bi9013209

Experimental method

X-RAY DIFFRACTION (2.3 Å)