3jqp
Crystal structure of the H286L mutant of Ferredoxin-NADP+ reductase from Plasmodium falciparum with 2'P-AMPCrystal structure of the H286L mutant of Ferredoxin-NADP+ reductase from Plasmodium falciparum with 2'P-AMP
Structural highlights
FunctionFENR_PLAF7 May play a role in the terminal step of the DOXP/MEP pathway for isoprenoid precursor biosynthesis.[1] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe 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. Plasmodium falciparum ferredoxin-NADP+ reductase His286 plays a dual role in NADP(H) binding and catalysis.,Crobu D, Canevari G, Milani M, Pandini V, Vanoni MA, Bolognesi M, Zanetti G, Aliverti A Biochemistry. 2009 Oct 13;48(40):9525-33. PMID:19736991[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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