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

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

Salta

Congreso; LV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) y de la Panamerican Society for Biochemistry and Molecular Biology; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) y de la Panamerican Society for Biochemistry and Molecular Biology

Ferredoxin-NADP+ reductases (FNRs) constitute a family of monomeric hydrophilic proteins that contain FAD as a prosthetic group. They areclassified as either plant- or mitochondrial-type FNRs. Plant-type FNRs are divided into plastidic and bacterial classes. Bacterial FNRs participatein metabolic pathways that are especially appropriate for the development of microbicidal agents because they are not present in humans. PlastidicFNRs have a conserved tyrosine residue at the carboxyl terminus which is interacting with FAD isoalloxazine. This residue would be displaced toallow the entry of NADP+. Plastidic FNRs show between 20- and 100-times greater exchange rates than bacterial enzymes. The latter, on the otherhand, 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 the NADP+ molecule interactswith three arginines (R144, R174, and R184) that would generate a strongly structured site with high affinity for the NADP+ substrate. These threeamino 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 ispresent in PeaFNR (R229); R144 corresponds to a proline (P199) and R184 to a tyrosine (Y240). We have found NADP+ tightly bound to theEcFPR. 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 nucleotidetightly bound to the P site (the one of higher affinity) would be released from it only after the nicotinamide of the incoming substrate interacts atthe 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 thesubstrate-binding mechanism could be different. The presence of NADP+ in the reaction medium only decreased the catalytic efficiency of wildtype EcFPR, indicating an inhibition by NADP+. NADP+ binding caused stabilization on wild type EcFPR but not on mutants or PeaFNR. Ourresults indicate that the high-affinity nucleotide binding is essential for the modulation of the catalytic activity of EcFPR. This phenomenon couldbe related to a general mechanism of activity regulation in bacterial enzymes.