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J Biol Chem, Vol. 275, Issue 2, 1471-1478, January 14, 2000
From the 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 The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF199509.
Cloning and Characterization of a Novel Human Dual Flavin
Reductase*
,,
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
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.
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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