The 2.1 Å resolution crystal structure of
flavin reductase P with the inhibitor nicotinamide adenine
dinucleotide (NAD) bound in the active site has been determined.
NAD adopts a novel, folded conformation in which the nicotinamide
and adenine rings stack in parallel with an inter-ring
distance of 3.6 Å. The pyrophosphate binds next to
the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine
moiety faces away from the flavin. The observed NAD conformation
is quite different from the extended conformations observed
in other enzyme/NAD(P) structures; however, it resembles
the conformation proposed for NAD in solution. The flavin
reductase P/NAD structure provides new information about
the conformational diversity of NAD, which is important
for understanding catalysis. This structure offers the
first crystallographic evidence of a folded NAD with ring
stacking, and it is the first enzyme structure containing
an FMN cofactor interacting with NAD(P). Analysis of the
structure suggests a possible dynamic mechanism underlying
NADPH substrate specificity and product release that involves
unfolding and folding of NADP(H).