DIFFERENTIAL NADP+ BINDING MODE IN BACTERIAL AND PLASTIDIC FERREDOXIN-NADP+ REDUCTASES

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

Salta

Congreso; The LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SOCIEDAD ARGENTINA DE INVESTIGACIÓN BIOQUÍMICA Y BIOLOGÍA MOLECULAR (SAIB) y otras

Ferredoxin-NADP+ reductases (FNR) constitute a family of monomeric hydrophilic proteins that contain FAD as a prosthetic group. They are classified as plant- and mitochondrial-type FNR. Plant-type FNR are divided into plastidic and bacterial classes.Bacterial FNR participate in metabolic pathways which are especially appropriate for the development of microbicidal agents because they are not present in humans.Plastidic FNR have a conserved tyrosine residue at the carboxyl terminus which is interacting with FAD isoalloxazine. This residue would be displaced to allow the entry of NADP+. Plastidic FNR show between 20 and 100 times greater exchange rates than bacterial enzymes. The latter, on the other hand, have a structured variable terminal carboxyl end that has not allowed to propose models justifying how the substrate reaches the active site.The crystallographic structure of bacterial Escherichia coli FNR (EcFPR) with the bound nucleotide shows that NADP+ molecule interacts with three arginines (R144, R174 and R184) that would generate a strongly structured site with high affinity for the NADP+ substrate. . These three amino acids are conserved in other bacterial FNR, but not in the highly efficient plastidic enzymes found in plant chloroplasts and cyanobacteria.The structural alignment of EcFPR with the plastidic Pisum sativum FNR (PeaFNR) shows that of these three arginines only R174 in EcFPR is present in PeaFNR (R229); R144 corresponds to a proline (P199) and R184 to a tyrosine (Y240). We have found NADP+tightly bound to the EcFPR. The bound nucleotide and the structured carboxyl terminus in bacterial enzymes could be the cause of their slower exchange rate.We propose a new model of catalysis for bacterial FNR in which NADP+ would interact with two different affinity sites (N and P). The nucleotide tightly bound to the P site (the one of higher affinity) would be released from it only after the nicotinamide of the incoming substrate interacts at the N site.We have cross-substituted EcFPR arginines with proline and tyrosine residues and replaced both amino acids with arginines in PeaFNR. We analyzed all proteins by kinetic, thermodynamic and stability studies.We found that the EcFPR mutants lost the ability to tightly bind NADP+ .Therefore the Arg mutations would be interfering with the NADP+ binding site. In PeaFNR mutants, NADP+ affinity was not affected thus, the substrate binding mechanism could be different.The presence of NADP+ in the reaction medium only decreased the catalytic efficiency of wild type EcFPR indicating an inhibition by NADP+.NADP+ binding caused stabilization on wild type EcFPR but not on mutants or PeaFNR. Our results indicate that the high-affinity nucleotide binding is essential for the modulation of the catalytic activity of EcFPR. This phenomenon could be related to a general mechanism of activity regulation in bacterial enzymes.