The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPH or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction 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 important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2 -OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.Flavins are well known as key prosthetic groups of a large number of redox enzymes named flavoproteins. More recently, protein-free flavins, riboflavin, FMN, or FAD, were also suggested to play, as electron transfer mediators, important biological functions, for example during ferric iron reduction (1-3), activation of ribonucleotide reductase (4, 5), bioluminescence (6, 7), and oxygen activation (8) (Scheme 1).The reduction of free flavins by reduced pyridine nucleotides NADPH or NADH is not an efficient reaction. The kinetics is slow unless very high nonphysiological concentrations of both reactants are present in the reaction mixture (8). As a consequence, living organisms have evolved enzymes that catalyze the reduction of riboflavin, FMN, and FAD by NADPH and NADH and are called NAD(P)H:flavin oxidoreductases or flavin reductases. It is now well established that such enzymes are present in all microorganisms, including the luminous marine bacteria, and also in mammals (1). A recent study has shown that flavin reductase activities are present in erythrocytes and in various human tissues (liver, heart, kidney, and lung) (9).In most cases, a single living organism contains multiple flavin reductases different in enzymatic nature and molecular mass. The luminous bacteria, Vibrio harveyi, contains at least three types of FMN reductases (10 -14). In Escherichia coli at least two flavin reductases have been isolated. One, named Fre, is a 26.2-kDa enzyme using both NADH and NADPH as electron donors (4), whereas the other is the sulfite reductase, a 780-kDa enzyme using NADPH exclusively (15). Still very little is known on the structure and the mechanisms of flavin reductases. No three-dimensional structure of such an enzyme is available yet, and only recently...

In order to link to the 5′-end of oligonucleotides the flavin analogs 9a,b possessing only one terminal hydroxy group on the side chain, the phosphoramidite and the H-phosphonate coupling methods were developed. Surprisingly, after reaction of compounds 9a,b with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, the flavin phosphoramidates 11a,b were isolated instead of the expected phosphoramidite derivatives 10a,b. A very efficient photooxidation process occurred probably during the isolation of the products. From the prepared flavin H-phosphonates 12a,b, flavin-thymine nucleotides and flavin-oligonucleotide adducts were synthesized for the first time. The versatility of the method was demonstrated in the oxidation step with the synthesis of the flavin-thymine nucleotides 15-17 possessing a phosphodiester, a phosphorothioate, and a methyl phosphate linkage, respectively. This method is of general interest with regard to the extensive research developed for preparing flavin analogs and modified oligonucleotides possessing interesting biological or/and catalytic properties.

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