Cloning and characterization of a novel human dual flavin reductase.
Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities 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 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescence in situ hybridization analysis. 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 cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochrome c, with apparent K(m) and k(cat) values of 21 microM and 1.3 s(-1), respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.
Paine
M J
MJ
Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom.
Garner
A P
AP
Powell
D
D
Sibbald
J
J
Sales
M
M
Pratt
N
N
Smith
T
T
Tew
D G
DG
Wolf
C R
CR
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms; D002874:Chromosome Mapping; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D003575:Cytochromes c1 / Q000378:metabolism; D038181:FMN Reductase; D005260:Female; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D003575:Cytochromes c1; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases