A different catalytic mechanism in bacterial Ferredoxin-NADP+ reductases due to a particular NADP+ binding mode

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

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Workshop; Third Meeting & First Workshop of the Argentine Network of Enzymatic Technology; 2021

Argentine Network of Enzymatic Technology

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant- and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes (FPRs) and the latter are also divided into two subclasses. Members of bacterial and plastidic classes differ not only in sequence alignment but also in the environment of the active site, FAD conformation and catalytic efficiency. Plastidic FNRs show turnover numbers between 20 and 100 times higher than FPRs and these differences have been related to their physiological functions. Moreover, FPRs contain a structured variable carboxyl terminus that has not allowed to propose logical models that justify how the substrate reaches the active site. FPRs participate in metabolic pathways especially appropriate for the development of antimicrobial agents since they are not present in humans. Thus, they can be used for the design of inhibitors in the fight against diseases caused by different pathogens.We have previously obtained crystals of a mutant Escherichia coli FNR (EcFPR) with bound NADP+, although the nucleotide substrate had not been added in the crystallization procedure. The NADP+ molecule interacts with three arginines (R144, R174 and R184) that are conserved in other FPRs of different subclasses, but not in the plastidic type enzymes. These residues could be responsible for generating a structured site with a very high affinity for NADP+.In this work, we used both an enzymatic method based on the reaction of glucose-6-phosphate dehydrogenase (G6PD) and HPLC to measure the nucleotide content in the reductases. We demonstrated that purified EcFPR contains tightly bound NADP+ at a ratio of 0.99 mol/mol. The nucleotide remains attached to the enzyme after extensive dialysis and gel filtration. In order to elucidate if this also occurs with other FPRs or FNRs, we analyzed the enzymes from Pectobacterium carotovorum (PcFPR) and Brucella abortus (BaFPR) and two plastidic type FNRs, from Pisum sativum (PeaFNR) and Leptospira interrogans (LepFNR). We observed that althought with different extent, all FPRs contain the nucleotide bound. In contrast, the plastidic type FNRs did not. We found a correlation between the amounts of NADP+ bound to the FNRs and their catalytic rate constants.By kinetic studies we observed that the presence of NADP+ considerably reduced the catalytic activity of EcFPR, which was recovered when the nucleotide was removed from the enzyme.Furthermore, we analyzed the effect of the bound NADP+ on the structural stability of FNRs by Thermal shift assays and showed that the NADP+ binding produced a structural stabilization of FPRs, but no effect was observed on the plastidic counterparts.To further investigate the participation of the abovementioned arginine residues in the tight binding of NADP+, we constructed different EcFPR and PeaFNR mutant enzymes. The structural and kinetic characterization of these proteins allowed us to confirm that R144 and R184 in EcFPR are involved in a structured site with a very high affinity for NADP+. The replacement of the residues present in PeaFNR in the analogous positions by arginines did not produce an increase in the content of bound NADP+ and no catalytic alterations were observed, suggesting that the NADP+ binding mode would be different in the plastidic FNR.Based on our observations we propose a novel catalytic and regulatory mechanism for the EcFPR in which a tightly bound NADP+ substrate itself controls this flavoenzyme activity and would be involved in the NADPH/NADP+ regulation. At low cellular NADPH concentration, the enzyme displays a basal activity as it is inhibited by the tightly bound NADP+. When the NADPH concentration increases, the oxidized nucleotide is released and the enzyme activated, thus accelerating the removal of the reduced nucleotide excess. This would be essential to ensure bacterial life by modulating the NADP(H) pool and consequently preserving the bacterial redox homeostasis. This phenomenon might be used as a differential target for the inactivation of metabolic pathways in which the FPRs participates in pathogenic bacteria.