Structure-function relationship of Vibrio harveyi NADPH-flavin oxidoreductase FRP: essential residues Lys167 and Arg15 for NADPH binding.
Vibrio harveyi NADPH-FMN oxidoreductase (FRP) catalyzes flavin reduction by NADPH. In comparing amino acid sequence and crystal structure with Escherichia coli NfsA, residues N134, R225, R133, K167, and R15 were targeted for investigation of their possible roles in the binding and utilization of the NADPH substrate. By mutation of each of these five residues to an alanine, steady-state rate analyses showed that the variants K167A and R15A had apparently greatly increased K(m,NADPH) and reduced k(cat)/K(m,NADPH), whereas little or much more modest changes were found for the other variants. The deuterium isotope effects (D)(V/K) for (4R)-[4-(2)H]-NADPH were markedly increased to 6.3 and 7.4 for K167A and R15A, respectively, indicating that the rate constants for NADPH and NADP(+) dissociation were greatly enhanced relative to the hydride transfer steps. Also, anaerobic stopped-flow analyses revealed that the equilibrium dissociation constant for NADPH binding (K(d)) to be 2.5-3.9 and 1.1 mM for K167A and R15A, respectively, much higher than the 0.4 μM K(d) for the native FRP, whereas the k(cat) of these two variants were similar to that of the wild-type enzyme. Moreover, the K167 to alanine mutation led to even a slight increase in k(cat)/K(m) for NADH. These results, taken together, provide a strong support to the conclusion that K167 and R15 each was critical in the binding of NADPH by FRP. Such a functional role may also exist for other FRP homologous proteins.
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Hae-Won
HW
Chung
Shiao-Chun
SC
Tu
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001120:Arginine; D003903:Deuterium / Q000737:chemistry; D038181:FMN Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014733:Vibrio / Q000201:enzymology*
D009249:NADP; D001120:Arginine; D003903:Deuterium; D038181:FMN Reductase; D008239:Lysine