Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
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Molecular Cloning and Expression of Fusion Proteins Containing Human Cytochrome P450 3As and Rat NADPH-P450 Reductase in Escherichia coli

  • Chun, Young-Jin (College of pharmacy, Chung-Ang University) ;
  • Guengerich, F-Peter (Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine)
  • Published : 2002.09.01
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Abstract

Cytochrome P450 3As such as 3A4 and 3A5 metabolize a wide range of pharmaceutical compounds. The vectors for the expression of fusion protein containing an N-terminal human P450 3A4 or P450 3A5 sequences and a C-terminal rat NADPH-cytochrome P450 reductase moiety were constructed. These plasmids were used to express the fusion protein in Escherichia coli DH5$\alpha$ cells. High levels of expression were achieved (100~200 nmol/liter) and the expressed fusion protein in E. coli membranes were catalytically active for nifedipine oxidation, a typical enzymatic activity of P450 3A4. The NADPH-P450 reductase activities of these fusion protein were also determined by measuring reduction of cytochrome c. To fine a specific Inhibitor of P450 3A4 from naturally occurring chemicals, a series of isothiocyanate compounds were evaluated for the inhibitory activity of P450 using the fusion proteins in E. coli membranes. Of the five isothiocyanates (phenethyl isothiocyanate, phenyl isothiocyanate, benzol isothiocyanate, benzoyl isothiocyanate and cyclohexyl isothiocyanate) tested, benzoyl isothiocyanate showed a strong inhibition of P450 3A4 with an $IC_{50}$value of 2.8 $\mu\textrm{M}$. Our results indicate that the self-sufficient fusion protein will be very useful tool to study the drug metabolism and benzyl isothiocyanate may be valuable for characterizing the enzymatic properties of P450 3A4.

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References

  1. Biochem. J. v.357 Nitric oxide synthases:structure, function and inhibition Alderton,W.K.;Cooper,C.E.;Knowles,R.G. https://doi.org/10.1042/0264-6021:3570593
  2. Arch. Biochem. Biophys. v.330 Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichta coli, purification, and catalytic properties of the enzyme in bacteial cells and after purification Chun,Y.J.;Shimada,T.;Guengerich,F.P. https://doi.org/10.1006/abbi.1996.0224
  3. Cancer Res. v.52 New potential chemopreventive agents for lung carcinogenesis of tobacco-specific nitrosamine Chung,F.L.;Morse,M.A.;Eklind,K.I.
  4. Arch. Biochem. Biophys. v.396 Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein Deeni,Y.Y.;Paine,M.J.;Ayrton,A.D.;Clarke,S.E.;Chenery,R.;Wolf,C.R. https://doi.org/10.1006/abbi.2001.2585
  5. Proc. Natl. Acad. Sci. USA v.89 High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein Fisher,C.W.;Shet,M.S.;Caudle,D.L.;Martin-Wixtrom,C.A.;Estabrook,R.W. https://doi.org/10.1073/pnas.89.22.10817
  6. Arch. Biochem. Biophys. v.305 Expression of modified human cytochrome p450 3Α4 in escherichia coli and purification and reconstitution of the enzyme Gillam,E.M.J.;Baba,T.;Kim,B.R.;Ohmori,S.;Guengerich,F.P. https://doi.org/10.1006/abbi.1993.1401
  7. Arch. Biochem. Biophys. v.317 Expression of cytochrome P450 3Α5 in Escherichia coli: effects of 5' modification, purification, spectral characterization, reconstitution conditions, and catalytic activities Gillam,E.M.J.;Gio,Z.;Ueng,Y.F.;Yamazaki,H.;Cock,I.;Reilly,P.E.;Hooper,W.D.;Guengerich,F.P. https://doi.org/10.1006/abbi.1995.1177
  8. Chem. Res. Toxicol. v.13 Inactivation of cytochrome P450 2Β1 by benzyl isothlocyanate, a chemopreventive agent from cruciferous vegetables Goosen,T.C.;Kent,U.M.;Brand,L.;Hollenberg,P.F. https://doi.org/10.1021/tx000133y
  9. J. Pharmacol. Exp. Ther. v.205 An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin Greelee,W.F.;Poland,A.
  10. J. Pharmacol. Exp. Ther. v.296 Effects of benzyl isothiocyanate on rat and human cytochromes P450: identification of metabolites formed by P450 2B1 Goosen,T.C.;Mills,D.E.;Hollenberg,P.F.
  11. Analysis and characterization of enzymes. Principles and Methods of Toxicology Guengerich,F.P.;Hayes,A.W.(ed.)
  12. Methods Enzymol v.272 Purification of functional recombinants P450s from bacteria Guengerich,F.P.;Martin,M.V.;Guo,Z.;Chun,Y.J. https://doi.org/10.1016/S0076-6879(96)72006-2
  13. Cancer Lett. v.150 Effects of phenethyl isothiocyanate and benzyl isothiocyanate, individually and in combination, on lung tumorigenesis induced in A/J mice by benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone Hecht,S.S.;Kenney,P.M.;Wang,M.;Trushin,M.;Upadhyaya,P. https://doi.org/10.1016/S0304-3835(99)00373-0
  14. The CYP3 Family. Cytochromes P450: Metabolic and Toxicological Aspects CRC Maurel,P.;Ioannides,C.(Ed.)
  15. Cancer Lett. v.62 Effect of frequency of isothiocyanate administration on inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary adenoma formation in A/J mice Morse,M.A.;Eklind,K.I.;Amin,S.G.;Chung,F.L. https://doi.org/10.1016/0304-3835(92)90201-6
  16. Trends Biochem. Sci. v.27 P450 BM3: the very model of a modern flavocytochrome Munro,A.W.;Leys,D.G.;McLean,K.J.;Marshall,K.R.;Ost,T.W.;Daff,S.;Miles,C.S.;Chapman,S.K.;Lys다,D.A.;Moser,C.C.;Page,C.C.;Dutton,P.L. https://doi.org/10.1016/S0968-0004(02)02086-8
  17. Drug Metab. Dispos. v.29 Inhibition and inactivation of human cytochrome P450 isoforms by phenethyl isothiocyanate Nakajima,M.;Yoshida,R.;Shimada,N.;Yamazaki,H.;Yokoi,T.
  18. J. Biol. Chem. v.239 The carbon-menoxide binding pigment of liver microsomes Omura,T.;Sato,R.
  19. Protein Expr. Purif. v.9 Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2:rat NADPH-cytochrome P450 reductase fusion protein Parikh,A;Guengerich,F.P. https://doi.org/10.1006/prep.1997.0721
  20. Arch. Biochem. Biophys. v.330 The omega-hydroxlyation of lauric acid: oxidation of 12-hydronlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase Shet,M.;Fisher,C.W.;Holmans,P.L.;Estabrook,R.W. https://doi.org/10.1006/abbi.1996.0243
  21. Proc. Natl. Acad. Sci. USA v.90 Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase Shet,M.;Fisher,C.W.;Holmans,P.L.;Estabrook,R.W. https://doi.org/10.1073/pnas.90.24.11748
  22. Cancer Res. v.51 Inhibitory effects of phenethyl isothiocyanate on N-nitrosobenzylmethylamine carcinogenesis in the rat esophagus Stoner,G.D.;Morrissey,D.T.;Heur,Y.H.;Daniel,E.M.;Galati,A.J.;Wagner,S.A.
  23. Carcinogenesis v.8 Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine or benzo[a]pyrene on pulmonary and forestomach neoplasia in A/J mice Wattenberg,L.W. https://doi.org/10.1093/carcin/8.12.1971
  24. Cancer Res. v.52 Inhibition of carcinogenesis by minor dietary constituents Wattenberg,L.W.
  25. Mol. Pharmacol. v.38 Studies on the expression and metabolic capabilities of human liver cytochrome P450ⅢA5(HLp3) Wrighton,S.A.;Brian,W.R.;Sari,M.A.;Iwasaki,M.;Guengerich,F.P.;Rarcy,J.L.;Molowa,D.T.;Vandenbranden,M.