Synthesis of a phosphorothioate analogue of flavin mononucleotide: reconstitution of the FMN-free form of NADPH-cytochrome P-450 reductase.
The chemical synthesis of riboflavin 5'-phosphorothioate (5'-FMNS) is described. 5'-FMNS is obtained from the alkaline hydrolysis of riboflavin 4',5'-cyclic phosphorothioate, which is produced upon reaction of riboflavin (RB) with thiophosphoryl chloride in trimethyl phosphate. 5'-FMNS has been tested for enzymatic reconstitution of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) depleted of its FMN prosthetic group, but containing its full complement (1 equiv) of FAD. The synthesis, purification, and characterization of 5'-FMNS are reported, and documentation of its efficacy in reconstituting the reductase by fluorometric and absorbance spectrophotometric measurements, as well as enzymatic activity, is presented. Data indicate that 5'-FMNS is totally competent in reconstituting NADPH-cytochrome c reductase activity, which requires the presence of both FAD and a flavin mononucleotide, and its fluorescence is completely quenched upon addition to FMN-free NADPH-cytochrome P-450 reductase.
D005486:Flavin Mononucleotide / Q000302:isolation & purification / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D011485:Protein Binding; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D007202:Indicators and Reagents; C054180:riboflavin 5'-phosphorothioate; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase; D012256:Riboflavin