CONICET | Buscador de Institutos y Recursos Humanos

Ferredoxin-NADP(H) reductases (FNRs, EC 1.18.1.2) constitute a family of hydrophilic, FAD-containing monomeric enzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin, heme-oxigenase) to redox-based metabolisms in plastids, mitochondria and bacteria. In heterotrophic bacteria, FNR activity provides reduced ferredoxin and flavodoxin to diverse reactions involved in amino acid and nucleotide metabolism, biotin synthesis, iron-sulfur cluster assembly and for the nitrogenase complex. Based on phylogenetic analysis the FNR variants present in most prokaryotes (collectively known as FPRs) have been classified into two subclasses represented by the Azotobacter vinelandii (subclass I) and the Escherichia coli (subclass II) FPR prototypes. Structures of bacterial and plastidic FNRs contain two distinct domains; the C-terminal domain that has a binding site for NADP(H) and the N-terminal region that binds the cofactor FAD. In bacterial enzymes, FAD is in bent conformation that is stabilised through an intramolecular H-bond between the N-6 atom of adenosine and the N-1 atom of isoalloxazine ring and an additional stacking interaction between an aromatic side chain in a carboxy-terminal extension and the adenosine moiety. The very low turnover rates for NADPH oxidation in diaphorase activity exhibited by bacterial FNRs with respect to those of plastidic enzymes could be due to the extended conformation of FAD in plastidic FNRs. Additionally, a subdivision of subclass I bacterial FNRs, IA and IB, was proposed based on differences in the primary sequences of their carboxy-terminal domains. To better understand the structural and functional divergence between subclass I FPRs, in the present work we have determined the crystal structure of XacFPR to a resolution of 1.5 Å. This FPR comes from the parasite Xanthomonas axopodis pv. citri, a Gram-negative bacterium responsible for citrus canker, a severe disease that affects most commercial citrus cultivars. The final structure reveals that XacFPR adopts many structural characteristics of this bacterial subclass although some structural differences among FPRs from subclass IA and IB have been detected. Thus, some loops distributed in the two domains show different mobility in terms of B factor values. Among the known structures of subclass IA FPRs, the side chain of the C-terminal glutamic residue (Glu258, numbering XacFPR) is engaged in an interaction with the O2 ribityl of FAD in the structures of Xac FPR and Pseudomonas aeruginosa FPR but not in Azotobacter vinelandii FPR. Looking at the FAD binding site, XacFPR presents in its structure a limited mobility of its phosphoadenosine motif because of an interaction through its phosphate with the side chain of Lys 259. These and other structural characteristics mainly centred in FAD environment and NADP+ binding site could help to understand better the kinetic behaviour of these oxidoreductases. Furthermore this enzyme represents a potential drug target to treat infections caused by Xanthomonas axonopodis pv. citri and thus a framework to develop new drugs against this disease.