KINETIC AND STRUCTURAL STUDY OF THE FERREDOXIN-NADP REDUCTASE FROM Xanthonomas

TONDO M.L; MUSUMECI M.A; ORELLANO E.G.; CECCARELLI E.A

Tucumán, Argentina

Congreso; SAIB 45th Annual Meeting; 2009

Sociedad Argentina de Investigación Bioquímica y Biología Moleculat (SAIB)

Ferredoxin-NADP(H) reductases (FNRs) are FAD-containing monomeric enzymes that deliver NADPH or low potential oneelectron donors (ferredoxin, flavodoxin, adrenodoxin) to redoxbased metabolisms in plastids, mitochondria and bacteria. The FNR variants present in most prokaryotes (FPRs) 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 possess a single fpr gene with the characteristic features of subclass I bacterial reductases. We have expressed in E. coli and purified to homogeneity the recombinant Xac FPR protein, rendering a monomeric product with an absorption spectrum characteristic of GB">flavin-containing proteins. Then, we further characterized Xac FPR protein by analyzing its kinetic behavior and interaction with the substrates NADPH, NADH, pea ferredoxin and E. coli flavodoxin. The accessibility of the prosthetic group FAD was also evaluated. Comparative analysis of the collected results with those reported for typical plastidic (i.e. pea) and bacterial (i.e. E. coli) FNRs suggest that Xac FPR resembles plastidic reductases regarding the interaction with nucleotidic substrates and the prosthetic group, but displays kinetic parameters typical of bacterial flavoenzymes.