TY - JOUR
T1 - Synthesis of a Phosphorothioate Analogue of Flavin Mononucleotide
T2 - Reconstitution of the FMN-Free Form of NADPH-Cytochrome P-450 Reductase
AU - Calhoun, Judith Porter
AU - Otvos, James D.
AU - Miziorko, Henry M.
AU - Krum, David P.
AU - Ugent, Steven
AU - Siler Masters, Bettie Sue
PY - 1987
Y1 - 1987
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0023664786&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023664786&partnerID=8YFLogxK
U2 - 10.1021/bi00391a019
DO - 10.1021/bi00391a019
M3 - Article
C2 - 3118943
AN - SCOPUS:0023664786
VL - 26
SP - 5344
EP - 5350
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 17
ER -