The mechanism and substrate specificity of the NADPH:flavin oxidoreductase from Escherichia coli.
The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPh or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2'-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.
Fieschi
F
F
Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, URA 332, CNRS, Université Joseph Fourier, Grenoble, France.
Nivière
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V
Frier
C
C
Décout
J L
JL
Fontecave
M
M
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D038181:FMN Reductase; D005415:Flavins / Q000378:metabolism / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010078:Oxazines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity
D005415:Flavins; D007202:Indicators and Reagents; D010078:Oxazines; D011994:Recombinant Proteins; D009243:NAD; C001559:7,8-dimethylalloxazine; D009249:NADP; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D006918:Hydroxyurea