IS THE NAD(P)H-FLAVIN OXIDOREDUCTASE FROM ESCHERICHIA-COLI A MEMBER OF THE FERREDOXIN-NADP(- EVIDENCE FOR THE CATALYTIC ROLE OF SERINE-49 RESIDUE() REDUCTASE FAMILY )

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a mon omer of 26.1 kDa which catalyzes the reduction of free flavins by NADP H 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 seq uence 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. k (cat) value of the S49A mutant was 35-fold lower than k(cat) of the wi ldtype enzyme. Determination of real K-d values for NADPH and lumichro me, 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 invo lved in the binding of the isoalloxazine ring. Moreover, the K-d 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, w hich has been shown to be essential for activity, is hydrogen-bonded t o 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.