CONICET | Buscador de Institutos y Recursos Humanos

Ferredoxin-NADP(H) reductases (FNRs) are FAD-containing monomeric enzymes that catalyze the reversible electron transfer between NADP(H) and the iron-sulfur protein ferredoxin (Fd) or FMN-containing flavodoxin (Fld). Based on their sequence and three-dimensional structures flavoenzymes with FNR activity can be grouped into two protein families referred to as plant-type ferredoxin reductases, harbouring a prototypic two-domain conformation, and glutathione reductase-type FNRs. The FNR variants present in most prokaryotes (called FPRs) have plant-type sequences and can be phylogenetically classified into two subclasses represented by the Azotobacter vinelandii (subclass I) and the Escherichia coli (subclass II) FPR prototypes. Xanthomonas axonopodis pv. citri (Xac) is a Gram-negative, aerobic bacteria that infects citrus plants. The genome of Xac has been recently sequenced and the fpr gene identified. In this study we analyzed the protein sequence of Xac FPR and found the six widely known clusters of highly conserved residues that define the structural family of plant-type FNRs. In addition we identified the characteristic features that defines this enzyme as a subclass I bacterial reductase. The fpr gene of Xac was cloned and expressed in E. coli and the recombinant protein was purified to homogeneity rendering a monomeric product with a molecular mass consistent with that predicted by the nucleotide sequence. The absorption spectrum of the purified Xac FPR protein was characteristic of flavin-containing proteins. The diaphorase activity with potassium ferricyanide as electron acceptor showed a reaction rate comparable to those reported for bacterial flavoenzymes, and 20-fold lower than the activities measured with the plant and cyanobacterial reductases. This results suggest that the fpr gene of Xac code for a functional protein with spectral and kinetic properties typical of bacterial flavoenzymes.

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