Catalytic mechanism and regulation of ferredoxin- NADP+ reductases from bacterial pathogens.

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

Paraná

Congreso; LIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

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

Ferredoxin-NADP+ reductases (FNR) constitute a family of proteins with a non-covalently bound FAD. They participate in redox metabolismscatalyzing the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. We identified that Escherichia coli FNR (EcFNR) ispurified with the substrate/product NADP+ tightly bound. This binding produces an activity inhibition which is lost when NADP+ is released.This inhibition would implicate a different catalytic mechanism than the one reported for the plastidic enzymes. The crystallographic structure ofEcFNR shows that NADP+ would interact with three arginine residues that could generate a site of very high affinity. These residues areconserved in other bacterial FNR but not in high efficiency plastidic enzymes from plants and cyanobacteria.We have studied the catalyticproperties of FNR from different pathogenic bacteria: E. coli, Pectobacteriumcarotovorum (PcFNR) and Brucella abortus (BaFNR). We foundthat PcFNR is purified with NADP+ tightly bound as EcFNR. BaFNR presented a small proportion of the nucleotide bound. Catalyticefficiencies were determined noting that they were similar between EcFPR and PcFPR, but it was higher in BaFPR. All of them were lower thanin plastidic FNR. Also, in EcFNR and PcFNR the absence of NADP+ decreased the stability of these proteins and the binding of NADP+ to theenzyme had a stabilizing effect on them.We propose that the high-affinity nucleotide binding is an essential catalytic and regulatory mechanismof these enzymes and it can be used as a differential target for the inactivation of metabolic pathways in which FNR participate in pathogenicbacteria.