  The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a
monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or
NADH as electron donor. The enzyme does not contain any prosthetic group but
accommodates both the reduced pyridine nucleotide and the flavin in a ternary
complex prior to oxidoreduction. The specificity of the flavin reductase for
the pyridine nucleotide was studied by steady-state kinetics using a variety of
NADP analogs. Both the nicotinamide ring and the adenosine part of the
substrate molecule have been found to be important for binding to the
polypeptide chain. However, in the case of NADPH, the 2'-phosphate group
destabilized almost completely the interaction with the adenosine moiety.
Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and
we have shown that both these molecules bind to the enzyme almost exclusively
by the nicotinamide ring. This provides evidence that the flavin reductase
exhibits a unique mode for recognition of the reduced pyridine nucleotide. In
addition, we have shown that the flavin reductase selectively transfers the
pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore
classified as an A-side-specific enzyme.
