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. 1992 Oct 1;287(Pt 1):195–200. doi: 10.1042/bj2870195

Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction. Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA.

J S Miles 1
PMCID: PMC1133143  PMID: 1417773

Abstract

1. Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance. 2. Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe. 3. A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774. This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium. 4. Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions. 5. The Sc. pombe cytochrome P-450 reductase gene was shown to contain no introns.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blondel A., Thillet J. A fast and convenient way to produce single stranded DNA from a phagemid. Nucleic Acids Res. 1991 Jan 11;19(1):181–181. doi: 10.1093/nar/19.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bredt D. S., Hwang P. M., Glatt C. E., Lowenstein C., Reed R. R., Snyder S. H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991 Jun 27;351(6329):714–718. doi: 10.1038/351714a0. [DOI] [PubMed] [Google Scholar]
  5. Crawford N. M., Smith M., Bellissimo D., Davis R. W. Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5006–5010. doi: 10.1073/pnas.85.14.5006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Haniu M., Iyanagi T., Miller P., Lee T. D., Shively J. E. Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Biochemistry. 1986 Dec 2;25(24):7906–7911. doi: 10.1021/bi00372a018. [DOI] [PubMed] [Google Scholar]
  9. Iyanagi T., Mason H. S. Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. Biochemistry. 1973 Jun 5;12(12):2297–2308. doi: 10.1021/bi00736a018. [DOI] [PubMed] [Google Scholar]
  10. Karplus P. A., Daniels M. J., Herriott J. R. Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science. 1991 Jan 4;251(4989):60–66. [PubMed] [Google Scholar]
  11. Katagiri M., Murakami H., Yabusaki Y., Sugiyama T., Okamoto M., Yamano T., Ohkawa H. Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA. J Biochem. 1986 Oct;100(4):945–954. doi: 10.1093/oxfordjournals.jbchem.a121807. [DOI] [PubMed] [Google Scholar]
  12. Käppeli O. Cytochromes P-450 of yeasts. Microbiol Rev. 1986 Sep;50(3):244–258. doi: 10.1128/mr.50.3.244-258.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mack D. H., Sninsky J. J. A sensitive method for the identification of uncharacterized viruses related to known virus groups: hepadnavirus model system. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6977–6981. doi: 10.1073/pnas.85.18.6977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Narhi L. O., Fulco A. J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem. 1986 Jun 5;261(16):7160–7169. [PubMed] [Google Scholar]
  15. Ostrowski J., Barber M. J., Rueger D. C., Miller B. E., Siegel L. M., Kredich N. M. Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase. J Biol Chem. 1989 Sep 25;264(27):15796–15808. [PubMed] [Google Scholar]
  16. PHILLIPS A. H., LANGDON R. G. Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J Biol Chem. 1962 Aug;237:2652–2660. [PubMed] [Google Scholar]
  17. Poole R. K., Lloyd D., Chance B. The development of cytochromes during the cell cycle of a glucose-repressed fission yeast, Schizosaccharomyces pombe 972h-. Biochem J. 1974 Feb;138(2):201–210. doi: 10.1042/bj1380201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Porter T. D. An unusual yet strongly conserved flavoprotein reductase in bacteria and mammals. Trends Biochem Sci. 1991 Apr;16(4):154–158. doi: 10.1016/0968-0004(91)90059-5. [DOI] [PubMed] [Google Scholar]
  19. Porter T. D., Beck T. W., Kasper C. B. NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein. Biochemistry. 1990 Oct 23;29(42):9814–9818. doi: 10.1021/bi00494a009. [DOI] [PubMed] [Google Scholar]
  20. Porter T. D., Kasper C. B. Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains. Proc Natl Acad Sci U S A. 1985 Feb;82(4):973–977. doi: 10.1073/pnas.82.4.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Porter T. D., Kasper C. B. NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins. Biochemistry. 1986 Apr 8;25(7):1682–1687. doi: 10.1021/bi00355a036. [DOI] [PubMed] [Google Scholar]
  22. Ruettinger R. T., Wen L. P., Fulco A. J. Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. J Biol Chem. 1989 Jul 5;264(19):10987–10995. [PubMed] [Google Scholar]
  23. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  25. Shen A. L., Porter T. D., Wilson T. E., Kasper C. B. Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis. J Biol Chem. 1989 May 5;264(13):7584–7589. [PubMed] [Google Scholar]
  26. Sutter T. R., Sanglard D., Loper J. C., Sangard D. Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins. J Biol Chem. 1990 Sep 25;265(27):16428–16436. [PubMed] [Google Scholar]
  27. Tomatsu S., Kobayashi Y., Fukumaki Y., Yubisui T., Orii T., Sakaki Y. The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene. Gene. 1989 Aug 15;80(2):353–361. doi: 10.1016/0378-1119(89)90299-0. [DOI] [PubMed] [Google Scholar]
  28. Urenjak J., Linder D., Lumper L. Structural comparison between the trout and mammalian hydrophilic domain of NADPH-cytochrome P-450 reductase. J Chromatogr. 1987 Jun 26;397:123–136. doi: 10.1016/s0021-9673(01)84995-5. [DOI] [PubMed] [Google Scholar]
  29. Watenpaugh K. D., Sieker L. C., Jensen L. H. The binding of riboflavin-5'-phosphate in a flavoprotein: flavodoxin at 2.0-Angstrom resolution. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3857–3860. doi: 10.1073/pnas.70.12.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yabusaki Y., Murakami H., Ohkawa H. Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene. J Biochem. 1988 Jun;103(6):1004–1010. doi: 10.1093/oxfordjournals.jbchem.a122370. [DOI] [PubMed] [Google Scholar]
  31. Yamano S., Aoyama T., McBride O. W., Hardwick J. P., Gelboin H. V., Gonzalez F. J. Human NADPH-P450 oxidoreductase: complementary DNA cloning, sequence and vaccinia virus-mediated expression and localization of the CYPOR gene to chromosome 7. Mol Pharmacol. 1989 Jul;36(1):83–88. [PubMed] [Google Scholar]

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