Biochemical characterization of ferredoxin-NADP+ reductase interaction with flavodoxin in Pseudomonas putida
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Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP+ reductase A (FprA) in Pseudomonas putida. Two residues (Phe256, Lys259) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala259 and FprA-Asp259 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-Ala259 and FprA-Asp259 were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe256 and Lys259 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. [BMB Reports 2012; 45(8): 476-481]
Abstract
Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP+ reductase A (FprA) in Pseudomonas putida. Two residues (Phe256, Lys259) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala259 and FprA-Asp259 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-Ala259 and FprA-Asp259 were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe256 and Lys259 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. [BMB Reports 2012; 45(8): 476-481]
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