Is the NAD(P)H:flavin oxidoreductase from Escherichia coli a member of the ferredoxin-NADP+ reductase family?. Evidence for the catalytic role of serine 49 residue.
The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.1 kDa which catalyzes the reduction of free flavins by NADPH or NADH. The flavin reductase Fre is the prototype of a new class of flavin reductases able to transfer electrons with no prosthetic group. It has been suggested that the flavin reductase could belong to the ferredoxin-NADP+ reductase (FNR) family, on the basis of limited sequence homologies. A sequence, conserved within the ferredoxin-NADP+ reductase family and present in the flavin reductase, is important for recognition of the isoalloxazine ring. Within this sequence, we have mutated serine 49 of the flavin reductase into alanine or threonine. kcat value of the S49A mutant was 35-fold lower than kcat of the wild-type enzyme. Determination of real Kd values for NADPH and lumichrome, a flavin analog, showed that recognition of the flavin is strongly affected by the S49A mutation, whereas affinity for the nicotinamide cofactor is only weakly modified. This suggests that serine 49 is involved in the binding of the isoalloxazine ring. Moreover, the Kd value for 5-deazariboflavin, in which the N-5 position of the isoalloxazine ring has been changed to a carbon atom, is not affected by the serine 49 to alanine mutation. This is consistent with the concept that the N-5 position is the main site for serine 49-flavin interaction. In the ferredoxin-NADP+ reductase family, the equivalent serine residue, which has been shown to be essential for activity, is hydrogen-bonded to the N-5 of the FAD cofactor. Taken together, these data provide the first experimental support to the hypothesis that the flavin reductase Fre may belong to the ferredoxin-NADP+ reductase family.
Nivière
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Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, UMR C5616, CNRS, Université Joseph Fourier, BP 53X, 38041 Grenoble Cedex 9, France.
Fieschi
F
F
Décout
J L
JL
Fontecave
M
M
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D038181:FMN Reductase; D005415:Flavins / Q000378:metabolism; D017665:Hydroxyl Radical; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics
D017931:DNA Primers; D005415:Flavins; D013912:Threonine; D017665:Hydroxyl Radical; D012694:Serine; D009249:NADP; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D000409:Alanine