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• W2018349053 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 fluorescencein 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 cytochromec, with apparent K m andk cat 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. 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 fluorescencein 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 cytochromec, with apparent K m andk cat 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. nitric-oxide synthase phosphate-buffered saline flavodoxin-NADP+ reductase fluorescent in situ hybridization polymerase chain reaction high performance liquid chromatography expressed sequence tag Tris-buffered saline with Tween 20 resuspension buffer 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.Dignam J.D. Strobel H.W. Biochem. Biophys. Res. Commun. 1975; 63: 845-852Crossref PubMed Scopus (127) Google Scholar, 2.Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar), followed by several other dual flavin enzymes including nitric-oxide synthases (NOS)1 in higher organisms (3.Bredt D.S. Hwang P.M. Glatt C. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar, 4.Schmidt H.H.W. Smith F.M. Nakane M. Murad F. Biochemistry. 1992; 31: 3243-3249Crossref PubMed Scopus (146) Google Scholar), and CYP102 (5.Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Abstract Full Text PDF PubMed Google Scholar) and sulfite reductase (6.Ostowski J. Barber M.J. Rueger D.C. Miller B.E. Siegel L.M. Kredich N.M. J. Biol. Chem. 1989; 264: 15796-15808Abstract Full Text PDF PubMed Google Scholar) in bacteria. More recently, the cDNA sequence encoding a putative FMN- and FAD-binding enzyme, methionine synthase reductase, has been described (7.Leclerc D. Wilson A. Dumas R. Gajuik C. Song D. Watkins D. Hing H.H.Q. Rommens J.M. Scherer S.W. Rosenblatt D.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3059-3069Crossref PubMed Scopus (353) Google Scholar). Cytochrome P-450 reductase, the most extensively characterized of these enzymes (8.Vermilion J. Coon M.J. J. Biol. Chem. 1978; 253: 8812-8819Abstract Full Text PDF PubMed Google Scholar, 9.Shen A.L. Kasper C.B. Schenkman J.B. Grein H. Cytochrome P-450. Springer-Verlag, New York1993: 35-59Google Scholar, 10.Backes W.L. Schenkman J.B. Greim H. Cytochrome P-450. Springer-Verlag, New York1993: 35-59Google Scholar), 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.Schacter B.A. Nelson E.B. Marver H.S. Masters B.S.S. J. Biol. Chem. 1972; 247: 3601-3607Abstract Full Text PDF PubMed Google Scholar), cytochrome b 5(12.Enoch H.G. Strittmatter P. J. Biol. Chem. 1981; 254: 8976-8981Abstract Full Text PDF Google Scholar), and the fatty acid elongation system (13.Ilan Z. Ilan R. Cinti D.C. J. Biol. Chem. 1981; 256: 10066-10072Abstract Full Text PDF PubMed Google Scholar), and can reduce cytochrome c (14.Williams C.H. Kamin H. J. Biol. Chem. 1962; 237: 587-595Abstract Full Text PDF PubMed Google Scholar). Both the crystal and NMR structure of the FMN domain of human cytochrome P-450 reductase (15.Zhao Q. Modi S. Smith G. Paine M.J.I. Wolf C.R. Tew D. Lian L-Y. Roberts G.C.K. Driessen H.P.C. Protein Sci. 1999; 8: 298-306Crossref PubMed Scopus (84) Google Scholar, 16.Barsukov I. Modi S. Lian L.-Y. Sze K.H. Paine M.J.I. Wolf C.R. Roberts G.C.K. J. Biomed. NMR. 1997; 10: 63-75Crossref PubMed Scopus (30) Google Scholar) and the crystal structure of the NH2-terminally truncated form of the rat enzyme (17.Wang M. Roberts D.C. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar) 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-terminal region shows similarities with the FAD-containing ferredoxin-NADP+reductases, thus leading to the hypothesis that cytochrome P-450 reductase has evolved as a fusion of these two ancestral proteins (18.Porter T.D. Trends Biochem. Sci. 1991; 16: 154-158Abstract Full Text PDF PubMed Scopus (116) Google Scholar,19.Smith G.C.M Tew D.G. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8710-8714Crossref PubMed Scopus (145) Google Scholar). A carboxyl-terminal cytochrome P-450 reductase-like domain is also a component of the NOS family of enzymes, where it is fused to an amino-terminal heme domain. The NOS reductase domain shuttles electrons from NADPH to the active site iron where the amino acid,l-arginine, is metabolized to nitric oxide (NO) (20.Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4158) Google Scholar). In addition to its normal physiological functions, cytochrome P-450 reductase plays a role in the reduction of one-electron acceptors such as the therapeutically important anticancer agents mitomycin c (22.Keyse S.R. Fracasso P.M. Heimbrook D.L. Rockwell S. Sligar S.G. Sartorelli A.C. Cancer Res. 1984; 44: 5638-5643PubMed Google Scholar), adriamycin (23.Bachur N.R. Gordon S.L. Gee M.V. Kon H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 954-957Crossref PubMed Scopus (584) Google Scholar), and the benzotriazine di-N-oxide, tirapazamine (24.Walton M.I. Wolf C.R. Workman P. Biochem. Pharmacol. 1992; 44: 251-259Crossref PubMed Scopus (84) Google Scholar). Evidence is also emerging that NOS can transfer electrons to these compounds via its reductase domain (25.Vásquez-Vivar J. Martasek P. Hogg N. Masters B.S.S. Pritchard K.A. Kalyanaraman B. Biochemistry. 1997; 36: 11293-11297Crossref PubMed Scopus (295) Google Scholar, 26.Garner A.P. Paine M.J.I. Rodriguez-Crespo I. Chinje E.C. Ortiz de Montellano P. Stratford I. Tew D. Wolf C.R.W. Cancer Res. 1999; 59: 1929-1934PubMed Google Scholar). The expression of these dual flavin reductases will therefore influence the outcome of cancer therapy. In this study we report the cloning of a novel member of the FNR family containing FMN and FAD as cofactors, which supports the NADPH-dependent metabolism of cytochrome c, the quinone anti-neoplastic agent doxorubicin, and menadione. Interestingly, the enzyme, which we have called NR1 (novelreductase 1) appears widely expressed in human cancer cell lines and, therefore, could play a potential role in the activation (or deactivation) of drugs used in cancer therapy. All chemicals were purchased from Sigma (Poole, Dorset, United Kingdom (UK)) and all enzymes and cell culture media were from Life Technologies, Inc. (Paisley, UK), except where stated. All solvents were of HPLC grade (Rathburn Chemicals Ltd., UK). The EST data base was screened for putative FAD- or FMN-containing proteins using the human P-450 reductase cDNA as the probe sequence (accession no. S90469). Two novel cDNAs were identified, one was subsequently reported to be methionine synthase reductase (7.Leclerc D. Wilson A. Dumas R. Gajuik C. Song D. Watkins D. Hing H.H.Q. Rommens J.M. Scherer S.W. Rosenblatt D.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3059-3069Crossref PubMed Scopus (353) Google Scholar) and the other (pNR1-SPORT) was used in these studies. This clone contained a 2506-nucleotide sequence and contained putative FAD- and FMN-binding domains. This cDNA in pSPORT (Life Technologies, Inc.) was used as a template for PCR amplification of the sequence for expression in baculovirus andEscherichia coli systems. Oligonucleotides 5′-GAGAATTCCATATGCCGAGCCCGCAGCTTCTG-3′ and 5′-GGAATTCCTCGAGTCAGGCCCACGTCTCTGTCTGGAA-3′ corresponding to putative 5′ and 3′ ends of the NR1 coding sequence were synthesized. The 5′ oligonucleotide contained overhanging NdeI andEcoRI restriction sites, while the 3′ oligonucleotide contained XhoI and EcoRI sites. Following 25 cycles of amplification using Pfu polymerase (Stratagene), the cDNA was ligated into pCR SCRIPT (Stratagene) to produce the plasmid pNR1-SCRIPT and sequenced to confirm that no PCR errors had been introduced. For baculoviral expression, the coding sequence was removed from pNR1-SCRIPT by EcoRI digestion and cloned into the unique EcoRI site of pFastBac Hta (Life Technologies, Inc.) and in frame with an amino-terminal 6-histidine linker and rTEV protease cleavage site. Recombinant baculovirus (vNR1) was produced following transposition of the cDNA sequence downstream of the polyhedrin promoter in Bacmid DNA and transfection into insect Sf9 cells using the Bac-to-Bac baculovirus expression system (Life Technologies, Inc.), following manufacturer's instructions. The FAD domain constructs were generated by PCR amplification and cloning of nucleotides 579–1860 (encoding amino acid residues 194–597). This region was cloned into pCRSCRIPTto produce pFAD-SCRIPT using a Stratagene kit system and sequenced to verify clone integrity. Forward (5′-GGGAATTCCATATGGTAGCTCACCCCGGCTCTCAGG-3′) and reverse (5′-GGAATTCCTCGAGTCAGGCCCACGTCTCTGTCTGCAA-3′) primers were then used, which contained NdeI and XhoI sites for subcloning into the unique NdeI/XhoI sites of pET15b (Novagen) downstream of a 6-histidine linker and thrombin cleavage site. The resulting plasmid pFAD-PET was used for expression of the FAD domain. For baculoviral expression of NR1, Sf9 cells were maintained at 27 °C in SF900 II media (Life Technologies, Inc.) according to standard procedures (27.0′Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman & Co., New York1992Google Scholar). For expression, a 300-ml suspension culture (∼2.0 × 106cells/ml) was infected with virus at a multiplicity of approximately 2 plaque-forming units/cell. Cells were harvested 72 h after infection and resuspended in 10 ml of PBS, 0.1% Tween 20. Protein purification steps were carried out at 4 °C. The suspension was sonicated (MSE probe, several short bursts at highest power) and centrifuged at 100,000 × g for 1 h (Sorvall Ultra Pro 80 with A641 rotor). The supernatant was loaded onto a 5-ml Hi-Trap nickel-agarose column (Amersham Pharmacia Biotech) and washed sequentially with 20 ml of 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10% glycerol; 20 ml Tris-HCl, pH 7.5, 500 mm NaCl, 60 mm imidazole, 10% glycerol; and then with 25 ml of PBS. Approximately 3 ml of yellow protein was then eluted with PBS,10% glycerol, 0.3 m imidazole. The protein was diluted with 10 ml of affinity buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1 mm dithiothreitol, 10% glycerol), and loaded onto a 2-ml 2′,5′-ADP-Sepharose column. The column was washed sequentially with 15 ml of affinity buffer and affinity buffer with 0.5 m NaCl. The protein was eluted in 2.5 ml of affinity buffer containing 0.5m NaCl and 10 mm 2′-AMP. FMN was added to 40 μm to replace any cofactor lost during purification, and protein exchanged into PBS, 10% glycerol using a PD-10 (Amersham Pharmacia Biotech) gel filtration column. Protein concentrations were determined by Bradford analysis using Bio-Rad reagents and bovine serum albumin as a protein standard. For E. coli expression of the FAD domain, BL21 (plys S) strains containing the domain expression plasmid pFAD-PET were grown overnight at 37 °C in LB broth containing ampicillin (50 μg/ml) and chloramphenicol (34 μg/ml). One-liter cultures of fresh LB broth were inoculated with 10 ml of the overnight culture and the bacteria grown at 37 °C to an optical density of 0.6–1.0. Isopropyl-1-thio-β-d-galactopyranoside was then added (0.5 mm) to initiate expression and the culture transferred to 30 °C and grown overnight. Cells were harvested at 5,000 ×g for 15 min and resuspended in 20 ml of binding buffer (5 mm imidazole, 500 mm NaCl, 20 mmTris-HCl, pH 8.0). The recombinant protein was extracted and purified over a 5-ml Hi-Trap nickel-agarose column (Amersham Pharmacia Biotech) as described (19.Smith G.C.M Tew D.G. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8710-8714Crossref PubMed Scopus (145) Google Scholar). The FAD domain was washed with binding buffer containing 60 mm imidazole and eluted with binding buffer containing 1 m imidazole. The protein eluted off the column was then exchanged into affinity buffer using PD-10 gel filtration columns and purified over 2′,5′-ADP-Sepharose as described above. Final yields of pure protein were between 2 and 5 mg/liter of culture. The final purified protein was stored in PBS, 10% glycerol at −20 °C. Antibodies against NR1 were generated in sheep using 1 mg of purified recombinant NR1-FAD domain by Scottish Antibodies Production Unit (Carluke, UK). Antibodies to full-length cytochrome P-450 reductase have been described previously (19.Smith G.C.M Tew D.G. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8710-8714Crossref PubMed Scopus (145) Google Scholar). SDS-polyacrylamide gel electrophoresis and immunoblots were carried out using a Mini-PROTEAN II (Bio-Rad) electrophoresis system. Except where indicated, proteins were separated using 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose (Schleicher & Schuell) according to manufacturer's instructions. For immunodetection, the blots were blocked in TBST (20 mmTris-HCl, pH 7.5, 0.5 m NaCl, 0.05% v/v Tween 20) with 5% w/v milk powder (Marvel) overnight at 4 °C with shaking. After washing several times with TBST, blots were incubated with appropriate antibody diluted in TBST, 5% milk powder at room temperature for 1–2 h. The binding of the primary antibodies was detected using a chemiluminescence detection system (Amersham Pharmacia Biotech, ECL). The secondary antibodies used were anti-rabbit IgG and anti-sheep IgG (Scottish Antibody Production Unit). The tumor cell lines MCF7, HepG2, HeLa, and NIH3T3 were cultured in Dulbecco's modified Eagle's medium. PEO1, EJ9, NCI H322, and HT29 cells were cultured in RPMI medium. All culture media were supplemented with 10% v/v heat-inactivated fetal calf serum, except for HepG2 cultures, which contained 15% v/v serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin, at 37 °C in 5% CO2. To prepare whole cell extracts, approximately 2 × 107cells were harvested by trypsinization, washed once with PBS, and frozen at −70 °C. Cells were resuspended in 1 ml of 100 mm PBS, 0.25 m sucrose and sonicated on ice using several pulses with an MSE probe. Following centrifugation at 4 °C, 12,000 × g for 1 h, the supernatant was aspirated and stored at −70 °C. For subcellular fractionation studies, approximately 2 × 108 MCF-7 cells were harvested, washed twice in PBS, and homogenized using a 20-ml glass homogenizer in 10 ml of resuspension buffer (RB) consisting of 0.25m sucrose, 50 mm HEPES, 1 mm EDTA, 0.2 mm dithiothreitol, 2 μg/ml aprotonin, 2 μg/ml leupeptin, 0.2 mm phenylmethylsulfonyl fluoride. Nuclear material and particulate cell matter were pelleted at 10,000 ×g for 10 min and resuspended in 5 ml of RB. The supernatant material was centrifuged at 100,000 × g for 1 h, and the resultant pellet was resuspended in 1 ml of RB. The supernatant, containing the cytoplasmic fraction, and the pellet, containing the membrane fractions, were stored at −70 °C. FMN and FAD content was determined by HPLC (28.Klatt P. Schmidt K. Werner E.R. Meyer B. Methods Enzymol. 1996; 268: 359-365Google Scholar) using a Hewlett Packard 1050 HPLC chromatograph and fluorescence detector. Flavins were released from NR1 by boiling for 5 min, and denatured protein removed by 20,000 ×g centrifugation for 10 min. FMN and FAD were detected by fluorescence (excitation, 450 nm; emission, 520 nm) following isocratic separation (10 mm sodium acetate, pH 6.0, methanol; v/v ratio 78:22) over a 25-cm Spherisorb ODS-2 5-μm column. Authentic FMN and FAD standards purchased from Sigma were used as control. Both were over 95% pure, as judged by HPLC analysis. Absorption spectra were obtained using a Shimadzu 160 UV spectrophotometer. Human Multiple Tissue Northern blots (CLONTECH) were hybridized with a 521-base pair cDNA fragment generated by SacI/SmaI restriction endonuclease digestion of pNR1. This probe was radiolabeled by incorporation of [32P]dCTP (RadPrime DNA labeling system, Life Technologies, Inc.) and purified using a Chroma Spin+ TE-30 column (CLONTECH). After a 1-h prehybridization, hybridization was carried out for 1 h using ExpressHyb buffer (CLONTECH) at 68 °C. The membrane was washed twice in 2× SSC, 0.05% sodium dodecyl sulfate for 20 min, twice in 0.1× SSC, 0.05% v/v sodium dodecyl sulfate at 50 °C for 20 min, and exposed to x-ray film at −70 °C with two intensifying screens. The full-length 2.6-kilobase NR1 cDNA segment was digested from pSPORT with EcoRI andHindIII and used as a probe in fluorescence in situ hybridization mapping (FISH). Standard cytogenetic techniques were used to prepare fixed normal lymphocyte slides. The probe was labeled with Spectrum Red dUTP using a nick translation kit (both Vysis, Downers Grove, IL) using the following kit protocol modifications. Slides were pretreated four times for 2 min in 2× SSC, pH 7.0, and dehydrated through 70%, 85%, and 100% ethanol (2 min each) prior to air drying. DNA was dried at 37 °C for 15 min, and an extra posthybridization wash of 1× SSC, 0.3% v/v Nonidet P-40 at 73 °C for 2 min was added between the other washes. Chromosomes were visualized using an Olympus BX60 fluorescent microscope fitted with a cooled CCD camera and using Vysis QUIPS image analysis software. Potassium ferricyanide and cytochromec reduction was measured as described for cytochrome P-450 reductase (29.Vermilion J.L. Ballou D.P. Massey V. Coon M.J. J. Biol. Chem. 1981; 256: 266-277Abstract Full Text PDF PubMed Google Scholar). The reduction of doxorubicin and menadione was carried out in 50 mm Tris-HCl (pH 7.5), 1 mm NADPH, and various substrate concentrations at 37 °C. The total incubation volume was 500 μl. Reactions were initiated by the addition of 10 μg of enzyme. The oxidation of NADPH was then monitored at 340 nm using a Shimadzu UV 2000 spectrophotometer. Final doxorubicin concentrations ranged from 20 to100 μm and menadione from 10 to 22.5 μm. Control reactions were carried out in the absence of active enzyme. A DNA fragment was identified, which contained an open reading frame with significant homology to human cytochrome P-450 reductase, following an extensive data base search of EST data base libraries. The cDNA insert for the EST was 2452 nucleotides in length (excluding the poly(A) tail) and contained the complete coding sequence for a putative cytochrome P-450 reductase-like enzyme (Fig.1). The initiation codon is predicted to be the first in-frame methionine residue based on sequence alignment with human cytochrome P-450 reductase and is preceded by several nucleotides bearing homology to the Kozak initiation sequence consensus (30.Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). There are also in-frame protein stop codons upstream of the predicted start site, which place this ATG codon in good context for the initiation of translation. A 1791-nucleotide sequence encodes a 597-amino acid residue polypeptide, NR1 (Fig. 1), with a predicted molecular mass of 66,700 Da. Comparative alignment of the identified amino acid sequence with human P-450 reductase and other FMN- and FAD-containing human flavoproteins shows sequence similarities ranging from 41% for NOS II and methionine synthase transferase to 44% for cytochrome P-450 reductase (Fig.2). The cytochrome P-450 reductase subfamily of enzymes contain distinctive amino-terminal FMN-binding and carboxyl-terminal FAD- and NADPH-binding domains, which are aligned for efficient electron transfer by a connecting domain (17.Wang M. Roberts D.C. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar). As shown in Fig. 2, a similar domain arrangement is found in NR1 with particularly strong sequence conservation in the regions shown to be involved in FMN, FAD, and NADPH cofactor binding. The major difference in domain organization is associated with the extreme amino-terminal region. Cytochrome P-450 reductase contains a hydrophobic 60-amino acid amino-terminal anchor domain, which is involved in tethering the molecule to the endoplasmic reticulum (9.Shen A.L. Kasper C.B. Schenkman J.B. Grein H. Cytochrome P-450. Springer-Verlag, New York1993: 35-59Google Scholar, 17.Wang M. Roberts D.C. Paschke R. Shea T.M. Masters B.S.S. Kim J.-J.P Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar). This domain is absent in NR1, implying a different cellular location for the enzyme. The 2.5-kb NR1 cDNA was used to probe a spread of human metaphase chromosomes. The gene was localized close to the telomere on the short arm of chromosome 9q34.3 by FISH analysis (Fig. 3). An 80-nucleotide gene sequence identical to NR1 cDNA has been identified in random mapping studies of chromosome 9 (31.Church D.M. Stotler C.J. Rutter J.L Murrell J.R. Troflatter J.A. Buckler A.J. Nat. Genet. 1994; 6: 98-105Crossref PubMed Scopus (270) Google Scholar), thus confirming the chromosomal localization. In order to determine whether the NR1 cDNA codes for a biologically active enzyme, we expressed the cloned cDNA using a baculovirus system. The full-length NR1 coding sequence was subcloned into pFastBac downstream of the very late polyhedron promoter, and the cDNA fused with a 6-histidine-tagged sequence at the amino terminus to facilitate affinity purification by nickel-agarose chromatography. Recombinant baculovirus vNR1 was generated by homologous recombination with Bacmid DNA. NR1 was detectable by polyacrylamide gel electrophoresis at around 3 days after infection, at which time cells were harvested for protein purification. Approximately 20% of recombinant enzyme remained in the soluble fraction following cell lysis and 100 × gcentrifugation. Recombinant NR1 was purified by affinity purification over nickel-agarose and ADP-Sepharose to a final purity of over 90% as judged by SDS-polyacrylamide gel electrophoresis (Fig.4 A). The final yield of purified NR1 was approximately 1.5 mg/liter suspension culture. Purified NR1 was yellow, indicating the presence of flavin, and bound to 2′,5′-ADP-Sepharose, indicating the presence of an NADPH-binding domain. HPLC analysis of heat-denatured enzyme determined that it released two fluorophores whose retention times exactly matched those of authentic FMN and FAD (Fig. 4 B). There were 1.2 and 1.1 mol each, respectively, of FMN and FAD bound per mole of enzyme. NR1 exhibited a UV-visible spectrum similar to cytochrome P-450 reductase (32.Vermilion J.L. Coon M.J. J. Biol. Chem. 1978; 253: 2694-2704Abstract Full Text PDF PubMed Google Scholar), possessing absorbance maxima at 380, 460, and 626 nm (Fig. 4 C). Like cytochrome P-450 reductase, the addition of NADPH under aerobic conditions caused a decrease in the absorbance at 380 and 460 nm, and an absorbance increase at 580 nm. Furthermore, the UV-visible spectrum of NR1 reduced with NADPH was stable over a 24-h period, which is consistent with the reduction of the flavin co-factors by NADPH and the production of an air-stable semiquinone form (10.Backes W.L. Schenkman J.B. Greim H. Cytochrome P-450. Springer-Verlag, New York1993: 35-59Google Scholar, 32.Vermilion J.L. Coon M.J. J. Biol. Chem. 1978; 253: 2694-2704Abstract Full Text PDF PubMed Google Scholar,46.Masters B.S.S. Bilimoria M.H. Kamin H. Gibson Q.H. J. Biol. Chem. 1965; 240: 4081-4088Abstract Full Text PDF PubMed Google Scholar). The above data showed that NR1 is a flavoenzyme that binds both FMN and FAD cofactors as predicted from the amino acid sequence. Furthermore, the spectral changes associated with the addition of NADPH indicate that electrons are transferred from NADPH to FAD and FMN, which indicates that NR1 follows the same pattern of electron transfer as in other dual flavin enzymes. The cytochrome P-450 reductase family of flavoenzymes are generally capable of reducing the hemoprotein cytochrome c, which thus serves as a model substrate for the comparative analysis of enzyme activity and electron transfer. Cytochrome c reducing activity was maximal when it occurred using potassium phosphate concentrations of between 300 and 400 mm and the enzyme had a pH optimum of around 8.0 (data not shown). There was also no detectable enzyme activity using NADH as a reducing cofactor. The conditions for optimal enzyme activity were thus similar to those observed for cytochrome P-450 reductase (9.Shen A.L. Kasper C.B. Schenkman J.B. Grein H. Cytochrome P-450. Springer-Verlag, New York1993: 35-59Google Scholar, 10.Backes W.L. Schenkman J.B. Greim H. Cytochrome P-450. Springer-Verlag, New York1993: 35-59Google Scholar). The kinetic parameters of cytochrome c reduction were compared with human cytochrome P-450 reductase. As shown in TableI, the apparent K mvalue of NR1 for cytochrome c was 21 μm, which was similar to cytochrome P-450 reductase (15 μm). Reported K m values of mammalian P-450 reductase for cytochrome c range between 5 and 21 μm (14.Williams C.H. Kamin H. J. Biol. Chem. 1962; 237: 587-595Abstract Full Text PDF PubMed Google Scholar,49.Philips A.H. Langdon R.G. J. Biol. Chem. 1962; 237: 2652-2660Abstract Full Text PDF PubMed Google Scholar, 50.Shen A. Kasper C.B. J. Biol. Chem. 1995; 270: 27475-27480Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The apparent k cat value was calculated as 1.3 s−1, which was approximately 100-fold lower than cytochrome P-450 reductase. NR1 also metabolized a range of one-electron acceptors, including the quinone-containing compounds doxorubicin and menadione (Table II). Although all the activities measured were significantly lower than cytochrome P-450 reductase (in the range 1–4%), they were reasonably similar to activities previously measured (25.Vásquez-Vivar J. Martasek P. Hogg N. Masters B.S.S. Pritchard K.A. Kalyanaraman B. Biochemistry. 1997; 36: 11293-11297Crossref PubMed Scopus (295) Google Scholar) for the reductase domain of NOS III (Table II). Taken together, these results indicate that the cloned cDNA encodes an authentic NADPH-dependent reductase enzyme, which is capable of catalyzing the reduction of cytochrome c and one-electron acceptors.Table IComparison of kinetic parameters of cytochrome c reduction by NR1 and cytochrome P-450 reductaseEnzymeK mV maxK catμmμmol/min/mgs −1NR121.3 ± 2.81.2 ± 0.11.3P-450 reductase15.2 ± 3.885.4 ± 13.8109.3Reactions contained 0.3 m potassium phosphate, pH 7.7, 50 μm NADPH, with varying amounts of cytochromec. Reactions were preincubated at 37 °C and initiated by the addition of NADPH. Values were determined by Lineweaver-Burke plot analysis and are the mean and standard deviation of three experiments." @default.
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• W2018349053 title "Cloning and Characterization of a Novel Human Dual Flavin Reductase" @default.
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• W2018349053 cites W126145571 @default.
• W2018349053 cites W1488511002 @default.
• W2018349053 cites W1497083865 @default.
• W2018349053 cites W1525040400 @default.
• W2018349053 cites W1531033444 @default.
• W2018349053 cites W1546570611 @default.
• W2018349053 cites W1554826669 @default.
• W2018349053 cites W1579297624 @default.
• W2018349053 cites W1579514744 @default.
• W2018349053 cites W1582769766 @default.
• W2018349053 cites W1594364959 @default.
• W2018349053 cites W1597669330 @default.
• W2018349053 cites W1639043875 @default.
• W2018349053 cites W1828405647 @default.
• W2018349053 cites W1834041853 @default.
• W2018349053 cites W192915975 @default.
• W2018349053 cites W1968068652 @default.
• W2018349053 cites W1976408994 @default.
• W2018349053 cites W1986080936 @default.
• W2018349053 cites W1987159760 @default.
• W2018349053 cites W1987322496 @default.
• W2018349053 cites W2004835079 @default.
• W2018349053 cites W2006369456 @default.
• W2018349053 cites W2006465601 @default.
• W2018349053 cites W2009013636 @default.
• W2018349053 cites W2028924242 @default.
• W2018349053 cites W2029587579 @default.
• W2018349053 cites W2034156530 @default.
• W2018349053 cites W2057469776 @default.
• W2018349053 cites W2061632173 @default.
• W2018349053 cites W2068906133 @default.
• W2018349053 cites W2076315300 @default.
• W2018349053 cites W2082207917 @default.
• W2018349053 cites W2082765051 @default.
• W2018349053 cites W2083233478 @default.
• W2018349053 cites W2094776852 @default.
• W2018349053 cites W2094978678 @default.
• W2018349053 cites W2100498403 @default.
• W2018349053 cites W2114000416 @default.
• W2018349053 cites W4889063 @default.
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