Molecular Characterization of FprB (Ferredoxin-NADP+ Reductase) inPseudomonas putida KT2440

Abstract

The fpr gene, which encodes a ferredoxin-NADP+reductase, is known to participate in the reversible redoxreactions between NADP+/NADPH and electron carriers,such as ferredoxin or flavodoxin. The role of Fpr and itsPseudomonas putida KT2440 onthe oxidative and osmotic stress responses has already beencharacterized [Le at al. (2006). Biochem. Biophys. Res.Comun . 339, 1246-1254]. In the genome of P. putidaKT2440, another Fpr homolog (FprB) has a 35.3% aminoacid identity with Fpr. The fprB gene was cloned andexpressed in Escherichia coli. The diaphorase activity assaywas conducted using purified FprB to identify the function ofFprB. In contrast to the fpr fprB wasnot affected by oxidative stress agents, such as paraquat,menadione, H2O2, and t-butyl hydroperoxide. However, ahigher level of fprB induction was observed under osmoticstress. Targeted disruption of fprB by homologous recombinationresulted in a growth defect under high osmotic conditions.Recovery of oxidatively damaged aconitase activity wasfaster for the fprB mutant than for the fpr mutant, yet stilsugest that the catalytic function of FprB may have evolvedto augment the function of Fpr in P. putida KT2440.