Expression, Purification and Characterization of the Ferredoxin-NADP(H) Reductase from the Phytopathogen Xanthomonas axonopodis pv. citri

TONDO, M.L.; OTTADO, J.; ORELLANO, E.G.

Flavins and Flavoproteins

S. Frago, C. Gómez-Moreno, M. Medina; University of Zaragoza

Lugar: Zaragoza; Año: 2008; p. 255 - 260

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 [1]. 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 [2]. 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 [3]. 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 [2]. This results suggest that the fpr gene of Xac code for a functional protein with spectral and kinetic properties typical of bacterial flavoenzymes.   [1] Aliverti, A., Pandini, V., Pennati, A., de Rosa, M. and Zanetti, G. (2008) Arch. Biochem. Biophys. doi: 10.1016/j.abb.2008.02.014. [2] Ceccarelli, E.A., Arakaki, A.K., Cortez, N. and Carrillo, N. (2004) Biochim. Biophys. Acta 1698, 155-165. [3] da Silva, A.C., Ferro, J.A., Reinach, F.C., Farah, C.S., Furlan, L.R., Quaggio, R.B., Monteiro-Vitorello, C.B., Van Sluys, M.A., Almeida, N.F., Alves, L.M. et al. (2002) Nature 417, 459-63.