The NAD(P)H : Flavin oxidoreductase from Escherichia coli - Evidence for anew mode of binding for reduced pyridine nucleotides

The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a mo nomer 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 redu ctase for the pyridine nucleotide was studied by steady-state kinetics usin g a variety of NADP analogs. Both the nicotinamide ring and the adenosine p art of the substrate molecule have been found to be important for binding t o the polypeptide chain. However, in the case of NADPH, the 2'-phosphate gr oup destabilized almost completely the interaction with the adenosine moiet y. Moreover, NADPH and NMNH are very good substrates for the flavin reducta se, and we have shown that both these molecules bind to the enzyme almost e xclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine n ucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide rin g and is therefore classified as an A-side-specific enzyme.