THE MECHANISM AND SUBSTRATE-SPECIFICITY OF THE NADPH-FLAVIN OXIDOREDUCTASE FROM ESCHERICHIA-COLI

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a mon omer of 26.2 kDa that catalyzes the reduction of free flavins by NADPH or NADH. Overexpression in E. coli now allows the preparation of larg e amounts of pure protein, Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-stat e kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for int er action with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an importan t role in that interaction, whereas the ribityl chain of the riboflavi n is not required for binding to the protein. On the other hand, the p resence of the 2'-OH of the ribityl chain stimulates the NADPH-depende nt reaction significantly, Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordere d mechanism in which NADPH binds first followed by riboflavin, Lumichr ome, a very good inhibitor of Fre, may be used to inhibit flavin reduc tase in E. coli growing cells. As a consequence, it can enhance the an tiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.