Vibrio harveyi NADPH:FMN oxidoreductase: preparation and characterization of the apoenzyme and monomer-dimer equilibrium.
A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with > or =80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 microM at 23 degrees C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme. 2-ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2-thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4 degrees C by equilibrium ultracentrifugation to undergo dimerization with dissociation constants of 1.8 and 3.3 microM, respectively.
Liu
M
M
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934, USA.
Lei
B
B
Ding
Q
Q
Lee
J C
JC
Tu
S C
SC
D001051:Apoenzymes / Q000737:chemistry; D038181:FMN Reductase; D007700:Kinetics; D046911:Macromolecular Substances; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D013379:Substrate Specificity; D014461:Ultracentrifugation; D014733:Vibrio / Q000201:enzymology*
D001051:Apoenzymes; D046911:Macromolecular Substances; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases