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Drug metabolism by Escherichia coli expressing human cytochromes P450

Abstract

The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.

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References

  1. Larson, J.R., Coon, M.J. and Porter, T.D. 1991. Alcohoi-inducible cytochrome P-450llE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli. J. Biol. Chem. 266: 7321–7324.

    CAS  PubMed  Google Scholar 

  2. Barnes, H.J., Arlotto, M.R. and Waterman, M.R. 1991. Expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 88: 5597–5601.

    Article  CAS  Google Scholar 

  3. Josephy, P.D., DeBruin, L.S., Lord, H.L., Oak, J., Evans, D.H., Guo, Z. et al. 1995. Bioactivation of aromatic amines by recombinant human cytochrome P450 1A2 expressed in bacteria: a substitute for mammalian tissue preparations in mutagenicity testing. Cancer Res. 55: 799–802.

    CAS  PubMed  Google Scholar 

  4. Guengerich, F.P. 1995. Human cytochrome P450 enzymes, pp. 473–535 in Cytochrome P450. Ortiz de Montellano, P.R. (ed.) Plenum Press, New York, NY.

    Chapter  Google Scholar 

  5. Jenkins, C.M. and Waterman, M.R. 1994. Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. J. Biol. Chem. 269: 27401–27408.

    CAS  PubMed  Google Scholar 

  6. Yamazaki, H., Ueng, Y.-F., Shimada, T. and Guengerich, P.P. 1995. Roles of divalent metal ions in oxidations catalyzed by recombinant cytochrome P450 3A4 and replacement of iron-sulfur proteins and oxygen surrogates. Biochemistry 34: 8380–8389.

    Article  CAS  Google Scholar 

  7. Murakami, H., Yabusaki, Y., Sakaki, T., Shibata, M. and Ohkawa, H. 1987. A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase. DNA 6: 189–197.

    Article  CAS  Google Scholar 

  8. Shet, M.S., Fisher, C.W., Holmans, P.L. and Estabrook, R.W. 1993. Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase. Proc. Natl. Acad. Sci. USA 90: 11748–11752.

    Article  CAS  Google Scholar 

  9. Chun, Y.-J., Shimada, T. and Guengerich, F.P. 1996. Construction of a human cytochrome P450 1A1: rat NADPH-P450 reductase fusion protein cDNA, expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification. Arch. Biochem. Biophys. 330: 48–58.

    Article  CAS  Google Scholar 

  10. Parikh, A. and Guengerich, F.P. 1997. Expression, purification,and characterization of a catalytically active human cytochrome P450 1A2: NADPH-cytochrome P450 reductase fusion protein. Protein Express. Purif. 9: 346–354.

    Article  CAS  Google Scholar 

  11. Metz, M.Z., Matsumoto, L., Winters, K.A., Doroshow, J.H. and Kane, S.E. 1996. Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene. Virology 217: 230–241.

    Article  CAS  Google Scholar 

  12. Lieschke, G.J., Rao, P.K., Gately, M.K. and Mulligan, R.C. 1997. Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo. Nature Biotechnology 15: 35–40.

    Article  CAS  Google Scholar 

  13. Shi, Y., Brown, E.D., and Walsh, C.T. 1994. Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions. Proc. Natl. Acad. Sci. USA 91: 2767–2771.

    Article  CAS  Google Scholar 

  14. Tarragona-Fiol, A., Taylorson, C.J., Ward, J.M. and Rabin, B.R. 1992. Production of mature bovine ribonuclease in Escherichia coli. Gene 118: 239–245.

    Article  CAS  Google Scholar 

  15. Dong, J. and Porter, T.D. 1996. Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli. Arch. Biochem. Biophys. 327: 254–259.

    Article  CAS  Google Scholar 

  16. Blake, J.A.R., Pritchard, M., Ding, S., Smith, G.C.M., Burchell, B., Wolf, C.R. and Friedberg, T. 1996. Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli. FEBS Lett. 397: 210–214.

    Article  CAS  Google Scholar 

  17. Shet, M.S., Fisher, C.W. and Estabrook, R.W. 1997. The function of recombinant cytochrome P450s in intact Escherichia coli cells: the I7α-hydroxylation of progesterone and pregnenolone by P450c17. Arch. Biochem. Biophys. 339: 218–225.

    Article  CAS  Google Scholar 

  18. Guengerich, P.P., Gillam, E.M.J. and Shimada, T. 1996. New applications of bacterial systems to problems in toxicology. Crit. Rev. Toxicol. 26: 551–583.

    Article  CAS  Google Scholar 

  19. Guengerich, F.P., Martin, M.V., Guo, Z. and Chun, Y.-J. 1996. Purification of recombinant human cytochrome P450 enzymes expressed in bacteria. Methods Enzymol. 272: 35–44.

    Article  CAS  Google Scholar 

  20. Omura, T. and Sato, R. 1964. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239: 2370–2378.

    CAS  PubMed  Google Scholar 

  21. Choc, M.G., Webster, D.A. and Caughey, W.S. 1982. Oxygenated intermediate and carbonyl species of cytochrome o (Vitreoscilla): characterization by infrared spectroscopy. J. Biol. Chem. 257: 865–869.

    CAS  PubMed  Google Scholar 

  22. Guengerich, F.P., Wang, P. and Mason, P.S. 1981. Immunological comparison of rat, rabbit, and human liver NADPH-cytochrome P-450 reductases. Biochemistry 20: 2379–2385.

    Article  CAS  Google Scholar 

  23. Porter, T.D., Wilson, T.E. and Kasper, C.B. 1987. Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli. Arch. Biochem. Biophys. 254: 353–367.

    Article  CAS  Google Scholar 

  24. Shen, A.L., Porter, T.D., Wilson, T.E. and Kasper, C.B. 1989. Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis. J. Biol. Chem. 264: 7584–7589.

    CAS  PubMed  Google Scholar 

  25. Schenkman, J.B., Remmer, H. and Estabrook, R.W. 1967. Spectral studies of drug interaction with hepatic microsomal cytochrome P-450. Mol. Pharmacol. 3: 113–123.

    CAS  PubMed  Google Scholar 

  26. Riley, M. and Labedan, B. 1996. Escherichia coli gene products: physiological functions and common ancestries, pp. 2118–2202 in Escherichia coli and Salmonella. Neidhardt, F.C. et al. (eds.) American Society of Microbiology, Washington, D.C.

    Google Scholar 

  27. Gold, L. 1991. Expression of heterologous proteins in Escherichia coli. Methods Enzymol. 185: 11–14.

    Article  Google Scholar 

  28. Olins, P.O. and Rangwala, S.H. 1990. Vector for enhanced translation of foreign genes in Escherichia coli. Methods Enzymol. 185, 115–119.

    Article  CAS  Google Scholar 

  29. Schoner, B.E., Belagaje, R.M. and Schoner, R.G. 1990. Enhanced translational efficiency with two-cistron expression system. Methods Enzymol. 185, 94–103.

    Article  CAS  Google Scholar 

  30. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  31. Burke, M.D. and Mayer, R.T. 1983. Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkylethers (alkoxyresorufins). Chem.-Biol. Interact. 45: 243–258.

    Article  CAS  Google Scholar 

  32. Burke, M.D. and Mayer, R.T. 1975. Inherent specificities of purified cytochromes P-450 and P-448 toward biphenyl hydroxylation and ethoxyresorufin deethylation. Drug Metab. Dispos. 3: 245–253.

    CAS  PubMed  Google Scholar 

  33. Distlerath, L.M., Reilly, P.E.B., Martin, M.V., Davis, G.G., Wilkinson, G.R. and Guengerich, P.P. 1985. Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem. 260: 9057–9067.

    CAS  PubMed  Google Scholar 

  34. Larrey, D., Distlerath, L.M., Dannan, G.A., Wilkinson, G.R. and Guengerich, F.P. 1984. Purification and characterization of the rat liver microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for genetic variation in oxidative drug metabolism. Biochemistry 23: 2787–2795.

    Article  CAS  Google Scholar 

  35. Knodell, R.G., Hall, S.D., Wilkinson, G.R. and Guengerich, F.P. 1987. Hepatic metabolism of tolbutamide: characterization of the form of cytochrome P-450 involved in methyl hydroxylation and relationship to in vivo disposition. J. Pharmacol. Exp. Ther. 241: 1112–1119.

    CAS  PubMed  Google Scholar 

  36. Gillam, E.M.J., Guo, Z., Martin, M.V., Jenkins, C.M. and Guengerich, F.P. 1995. Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch. Biochem. Biophys. 319: 540–550.

    Article  CAS  Google Scholar 

  37. Boddupalli, S.S., Estabrook, R.W. and Peterson, J.A. 1990. Fatty acid monooxygenation by cytochrome P-450BM-3. J. Biol. Chem. 265: 4233–4239.

    CAS  PubMed  Google Scholar 

  38. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  Google Scholar 

  39. Guengerich, F.P. 1994. Analysis and characterization of enzymes, pp 1259–1313 in Principles and Methods of Toxicology. Hayes, AW. (ed.) Raven Press, Ltd., New York, NY.

    Google Scholar 

  40. Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. 1981. Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 118: 197–203.

    Article  CAS  Google Scholar 

  41. Yasukochi, Y. and Masters, B.S.S. 1976. Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 251: 5337–5344.

    CAS  PubMed  Google Scholar 

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Correspondence to F. Peter Guengerich.

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Parikh, A., Gillam, E. & Guengerich, F. Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat Biotechnol 15, 784–788 (1997). https://doi.org/10.1038/nbt0897-784

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