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J Biol Chem, Vol. 275, Issue 2, 1471-1478, January 14, 2000

Cloning and Characterization of a Novel Human Dual Flavin Reductase*

Mark J. I. PaineDagger , Andrew P. GarnerDagger , David Powell§, Jennifer Sibbald, Mark Sales, Norman Pratt, Trudi Smith§, David G. Tew§, and C. Roland WolfDagger par

From the Dagger  Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom, § SmithKline Beecham Research, King of Prussia, Pennsylvania 19406-0939, and the  Cytogenetics Department, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom

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 Km and kcat values of 21 µM 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.

* This work was supported by SmithKline Beecham and United Kingdom Medical Research Council Grant G9203175.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF199509.

par To whom correspondence should be addressed. Tel.: 44-1382-632-621; Fax: 44-1382-668278; E-mail: rooney@dundee.ac.uk.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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