Cloning and Characterization of a Novel Human Dual Flavin Reductase

Abstract

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 M 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.Flavin-containing enzymes catalyze a broad spectrum of biochemical reactions ranging from oxidase, dehydrogenase, and mono-oxygenase reactions. Most flavoproteins contain either FMN or FAD as prosthetic groups; however, a small number of enzymes contain both cofactors. In mammalian systems, NADPH cytochrome P-450 oxidoreductase (cytochrome P-450 reductase) was the first such enzyme isolated (1, 2), followed by several other dual flavin enzymes including nitric-oxide synthases (NOS) 1 in higher organisms (3, 4), and CYP102 (5) and sulfite reductase (6) in bacteria. More recently, the cDNA sequence encoding a putative FMN-and FAD-binding enzyme, methionine synthase reductase, has been described (7). Cytochrome P-450 reductase, the most extensively characterized of these enzymes (8 -10), is found in the endoplasmic reticulum of most eukaryote cells and is an integral component of the monooxygenase system transferring electrons from NADPH to cytochromes P-450 via FMN and FAD co-factors. Cytochrome P-450 reductase may also donate electrons to heme oxygenase (11), cytochrome b 5 (12), and the fatty acid elongation system (13), and can reduce cytochrome c (14). Both the crystal and NMR structure of the FMN domain of human cytochrome P-450 reductase (15, 16) and the crystal structure of the NH 2 -terminally truncated form of the rat enzyme (17) have been resolved, providing high resolution structural information on this enzyme class. The amino-terminal region of cytochrome P-450 reductase bears striking amino acid homology with FMN-containing flavodoxins, while the carboxyl-term...

Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation

“…It is 38% identical to the human microsomal cytochrome P-450 reductase and is predicted to have an N-terminal FMN-binding domain and a C-terminal FAD/ NADPH-binding domain separated by a linker region. It is 41% identical to NR1, a soluble dual flavoprotein of unknown function (28). The predicted multidomainal organization of methionine synthase reductase is also suggested by its proteolytic lability during purification that results in the generation of two fragments (not shown) and contributes to the low yield following the limited proteolysis step (Table I).…”

“…The cytochrome c reductase activity of methionine synthase reductase (V max ϭ 0.44 mol min Ϫ1 mg Ϫ1 protein) is, however, significantly lower (1%) than that of cytochrome P-450 reductase but comparable with that of a newly described and homologous flavoprotein of unknown function, NR1 (28). DISCUSSION Functional deficiency of methionine synthase is inherited as an autosomal recessive disorder.…”

“…The latter is one of a small number of dual flavoproteins, a family that includes cytochrome P-450 reductase (22), nitric oxide synthase (38,39), sulfite reductase (40), and NR1 (28). It is 38% identical to the human microsomal cytochrome P-450 reductase and is predicted to have an N-terminal FMN-binding domain and a C-terminal FAD/ NADPH-binding domain separated by a linker region.…”

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

“…They are dimeric flavohaem enzymes : each monomer comprises a C-terminal diflavin reductase domain and an N-terminal oxygenase domain [4][5][6][7]. The reductase domain contains one mole equivalent of FAD and FMN, binds NADPH and is related structurally and functionally to cytochrome P450 reductase (CPR) [8,9], methionine synthase reductase (' MSR ' [10]) and protein NR1 [11]. The N-terminal oxygenase domain of NOS contains one mole equivalent of haem and possesses binding sites for L-arginine and (6R)-5,6,7,8-tetrahydrobiopterin (BH % [12]).…”

Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase

“…In S. enterica, the Fre protein was reported to catalyse the production of reduced flavin nucleotides which, in turn, reduce cob(III)alamin chemically to cob(II)alamin (Fonseca & Escalante-Semerena, 2000). The E. coli FldA protein, the human methionine synthase reductase (MSR) and the human novel reductase 1 (NR1) were shown to reduce cob(II)alamin to cob(I)alamin for the reductive activation of methionine synthase (Fujii et al, 1977;Olteanu & Banerjee, 2003;Paine et al, 2000;Wilson et al, 1999). In addition, the FldA protein in combination with the Fpr protein was reported to reduce cob(III)alamin to cob(I)alamin for AdoCbl production by the CobA adenosyltransferase (Fonseca & EscalanteSemerena, 2001).…”