• BMB Reports
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• v.45 no.8
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• pp.476-481
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• 2012

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP$^+$ reductase A (FprA) in Pseudomonas putida. Two residues ($Phe^{256}$, $Lys^{259}$) of FprA are likely to be important for interacting with Fld based on homology modeling. Site-directed mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-$Ala^{259}$ and FprA-$Asp^{259}$ proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-$Ala^{259}$ and FprA-$Asp^{259}$ were examined by isothermal titration calorimetry. We demonstrated, for the first time, that $Phe^{256}$ and $Lys^{259}$ are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1504-1512
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• 2007

The fpr gene, which encodes a ferredoxin-$NADP^+$ reductase, is known to participate in the reversible redox reactions between $NADP^+$/NADPH and electron carriers, such as ferredoxin or flavodoxin. The role of Fpr and its regulatory protein, FinR, in Pseudomonas putida KT2440 on the oxidative and osmotic stress responses has already been characterized [Lee at al. (2006). Biochem. Biophys. Res. Commun. 339, 1246-1254]. In the genome of P. putida KT2440, another Fpr homolog (FprB) has a 35.3% amino acid identity with Fpr. The fprB gene was cloned and expressed in Escherichia coli. The diaphorase activity assay was conducted using purified FprB to identify the function of FprB. In contrast to the fpr gene, the induction of fprB was not affected by oxidative stress agents, such as paraquat, menadione, $H_2O_2$, and t-butyl hydroperoxide. However, a higher level of fprB induction was observed under osmotic stress. Targeted disruption of fprB by homologous recombination resulted in a growth defect under high osmotic conditions. Recovery of oxidatively damaged aconitase activity was faster for the fprB mutant than for the fpr mutant, yet still slower than that for the wild type. Therefore, these data suggest that the catalytic function of FprB may have evolved to augment the function of Fpr in P. putida KT2440.