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...

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.