Insights in the NADP+ binding mode of bacterial ferredoxin- NADP+ reductases

CATALANO DUPUY, DANIELA L; MONCHIETTI, PAULA; CECCARELLI, EDUARDO A

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Congreso; LVI SAIB Meeting and XV SAMIGE Meeting; 2020

Argentine Society for Biochemistry and Molecular Biology (SAIB)

Ferredoxin-NADP+ reductases (FNR) constitute a family of proteins with a non-covalently bound FAD as a prosthetic group. They participate in redox metabolisms catalyzing the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant- and mitochondrial-type FNR. Plant-type FNR are divided into plastidic and bacterial classes. The plastidic FNR show between 20- and 100-times higher exchange rates than bacterial enzymes. We have obtained experimental evidence that Escherichia coli FNR (EcFPR) contains its NADP+ substrate tightly bound after isolation. The three-dimensional structure evidenced that NADP+ interacts with three arginines (R144, R174 and R184) which could generate a site of very-high affinity and great structuring. These arginines are conserved in other bacterial FNR but not in highly efficient plastidic enzymes. Based on a structural alignment, we have cross-substituted EcFPR arginines with proline and tyrosine residues, which are present in analogous positions in the plastidic Pisum sativum FNR (PsFNR) (P199 and Y240) and replaced both amino acids with arginines in PsFNR. We analyzed all proteins by kinetic, structural, thermodynamic and stability studies.We discovered that, while EcFPR contains tightly bound NADP+, its mutants lost the ability to bind the nucleotide, suggesting that mutations in Arg interfere with the NADP+ binding site. Moreover, the mutants showed significantly increased KM for NADP+ and lower catalytic efficiencies than the wild-type enzyme. The activity of EcFPR was inhibited by NADP+ but this behavior disappeared as arginines were removed. Unfolding studies showed that NADP+ binding stabilized EcFPR structure. By using DMAP as analog of the nicotinamide portion of NADP+ we observed an activation of EcFPR probably by releasing the tightly bound NADP+ from the enzyme. On the other hand, we observed PsFNR did not bind NADP+ and that the introduction of arginines in the PsFNR mutants was not enough to restore the bacterial NADP+ binding site. This difference was further evidenced by the absence of any effect over kinetic parameters or structure stability by NADP+ binding. Our studies indicate that the nucleotide binding characteristics between bacterial and plastidic FNR would be different and probably might be related with the differential catalytic efficiency observed. We propose that the high-affinity nucleotide binding is an essential catalytic and regulatory mechanism of bacterial FNR involved in redox homeostasis. This phenomenon might be used as a differential target for the inactivation of metabolic pathways in which the FNR participates in pathogenic bacteria.