Cloning and characterization of a novel human dual flavin reductase

Flavoprotein reductases play a key role in electron transfer in many physio logical processes. We have isolated a cDNA with strong sequence similaritie s to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilob ase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gen e was mapped to chromosome 9q34.3 by fluorescence in situ hybridization ana lysis. The amino acid sequence of the protein contained distinct FMN-, FAD- , and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cell s and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 62 6 nm. The purified enzyme reduced cytochrome c, with apparent K-m and k(cat ) values of 21 mu M and 1.3 s(-1), respectively, and metabolized the one-el ectron accepters doxorubicin, menadione, and potassium ferricyanide. Immuno blot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of h uman cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and oth er chemicals activated by one-electron reduction.