Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Self-Sufficient Catalytic System of Human Cytochrome P450 4A11 and NADPH-P450 Reductase

  • Han, Song-Hee (Department of Biological Sciences, Konkuk University) ;
  • Eun, Chang-Yong (Department of Biological Sciences, Konkuk University) ;
  • Han, Jung-Soo (Department of Biological Sciences, Konkuk University) ;
  • Chun, Young-Jin (College of Pharmacy, Chung-Ang University) ;
  • Kim, Dong-Hyun (School of Biological Sciences and Technology, Chonnam National University) ;
  • Yun, Chul-Ho (School of Biological Sciences and Technology, Chonnam National University) ;
  • Kim, Dong-Hak (Department of Biological Sciences, Konkuk University)
  • Published : 2009.04.30
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Abstract

The human cytochrome P450 4A11 is the major monooxygenase to oxidize the fatty acids and arachidonic acid. The production of 20-hydroxyeicosatetraenoic acid by P450 4A11 has been implicated in the regulation of vascular tone and blood pressure. Oxidation reaction by P450 4A11 requires its reduction partners, NADPH-P450 reductase (NPR). We report the functional expression in Escherichia coli of bicistronic constructs consisting of P450 4A11 encoded by the first cistron and the electron donor protein, NPR by the second. Typical P450 expression levels of wild type and several N-terminal modified mutants was observed in culture media and prepared membrane fractions. The expression of functional NPR in the constructed P450 4A11: NPR bicistronic system was clearly verified by reduction of nitroblue tetrazolium. Membrane preparation containing P450 4A11 and NPR efficiently oxidized lauric acid mainly to $\omega$-hydroxylauric acid. Bicistronic coexpression of P450 4A11 and NPR in E. coli cells can be extended toward identification of novel drug metabolites or therapeutic agents involved in P450 4A11 dependent signal pathways.

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References

  1. Baer, B. R. and Rettie, A. E. (2006). CYP4B1: an enigmatic P450 at the interface between xenobiotic and endobiotic metabolism. Drug Metab. Rev. 38, 451-476 https://doi.org/10.1080/03602530600688503
  2. CaJacob, C. A., Chan, W. K., Shephard, E. and Ortiz de Montellano, P. R. (1988). The catalytic site of rat hepatic lauric acid omega-hydroxylase. Protein versus prosthetic heme alkylation in the omega-hydroxylation of acetylenic fatty acids. J. Biol. Chem. 263, 18640-18649
  3. Capdevila, J. H., Falck, J. R. and Harris, R. C. (2000). Cytochrome P450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase. J. Lipid Res. 41, 163-181
  4. Chae, A. R., Shim, J. H. and Chun, Y. J. (2008). Mechanism of inhibition of human cytochrome P450 IAI and IBI by piceatannol. Biomol. Ther. 16, 336-342 https://doi.org/10.4062/biomolther.2008.16.4.336
  5. Clark, B. J. and Waterman, M. R. (1992). Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence. J. Biol. Chem. 267, 24568-24574
  6. Dong, M. S., Bell, L. C., Guo, Z., Phillips, D. R., Blair, I. A. and Guengerich, F. P. (1996). Identification of retained N-formylmethionine in bacterial recombinant mammalian cytochrome P450 proteins with the N-terminal sequence MALLLAVFL...: roles of residues 3-5 in retention and membrane topology. Biochemistry 35, 10031-10040 https://doi.org/10.1021/bi960873z
  7. Gillam, E. M., 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 https://doi.org/10.1006/abbi.1995.1329
  8. Guengerich, F. P. (1993). Cytochrome P450 enzymes. American Scientist 81, 440-447
  9. Gungerich, F. P. (2001). Analysis and characterization of enzymes. In Principles and Methods of Toxicology, 4th ed. (A. W. Hayes, Ed.), pp 1625-1687. Taylor & Francis, Philadephila
  10. Guengerich, F. P. (2002). Cytochrome P450 enzymes in the generation of commercial products. Nature Rev. Drug Discov. 1, 359-366 https://doi.org/10.1038/nrd792
  11. Guengerich, F. P. (2008). Cytochrome p450 and chemical toxicology. Chem. Res. Toxicol. 21, 70-83 https://doi.org/10.1021/tx700079z
  12. Guengerich, F. P., Gillam, E. M. J. and Shimada, T. (1996). New applications of bacterial systems to problems in toxicology. CRC Critical Reviews in Toxicology 26, 551-583 https://doi.org/10.3109/10408449609037477
  13. Hoch, U., Falck, J. R. and Ortiz de Montellano, P. R. (2000). Molecular basis for the omega-regiospecificity of the CYP4A2 and CYP4A3 fatty acid hydroxylases. J. Biol. Chem. 275, 26952-26958
  14. Hoch, U. and Ortiz De Montellano, P. R. (2001). Covalently linked heme in cytochrome p4504a fatty acid hydroxylases. J. Biol. Chem. 276, 11339-11346 https://doi.org/10.1074/jbc.M009969200
  15. Hoch, U., Zhang, Z., Kroetz, D. L. and Ortiz de Montellano, P. R. (2000). Structural determination of the substrate specificities and regioselectivities of the rat and human fatty acid omega-hydroxylases. Arch. Biochem. Biophys. 373, 63-71 https://doi.org/10.1006/abbi.1999.1504
  16. Hsu, M. H., Savas, U., Griffin, K. J. and Johnson, E. F. (2007). Human cytochrome p450 family 4 enzymes: function, genetic variation and regulation. Drug Metab. Rev. 39, 515-538 https://doi.org/10.1080/03602530701468573
  17. Imig, J. D. and Navar, L. G. (1996). Afferent arteriolar response to arachidonic acid: involvement of metabolic pathways. Am. J. Physiol. 271, F87-F93
  18. Kim, D., Cryle, M. J., De Voss, J. J. and Ortiz de Montellano, P. R. (2007). Functional expression and characterization of cytochrome P450 52A21 from Candida albicans. Arch. Biochem. Biophys. 464, 213-220 https://doi.org/10.1016/j.abb.2007.02.032
  19. Kim, D. and Guengerich, F. P. (2004). Selection of human cytochrome P450 1A2 mutants with enhanced catalytic activity for heterocyclic amine N-hydroxylation. Biochemistry 43, 981-988 https://doi.org/10.1021/bi035593f
  20. LeBrun, L. A., Hoch, U. and Ortiz de Montellano, P. R. (2002). Autocatalytic mechanism and consequences of covalent heme attachment in the cytochrome P4504A family. J. Biol. Chem. 277, 12755-12761 https://doi.org/10.1074/jbc.M112155200
  21. Ma, Y. H., Gebremedhin, D., Schwartzman, M. L., Falck, J. R., Clark, J. E., Masters, B. S., Harder, D. R. and Roman, R. J. (1993). 20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries. Circ. Res. 72, 126-136 https://doi.org/10.1161/01.RES.72.1.126
  22. Murphy, T. M., Vu, H. and Nguyen, T. (1998). The superoxide synthases of rose cells. Plant Physiol. 117, 1301-1305 https://doi.org/10.1104/pp.117.4.1301
  23. Ortiz de Montellano, P. R. (2005). In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., Ed.) Plenum Press, New York
  24. Ortiz de Montellano, P. R. (2008). Mechanism and role of covalent heme binding in the CYP4 family of P450 enzymes and the mammalian peroxidases. Drug Metab. Rev. 40, 405-426 https://doi.org/10.1080/03602530802186439
  25. Parikh, A., Gillam, E. M. J. and Guengerich, F. P. (1997). Drug metabolism by Escherichia coli expressing human cytochromes P450. Nature Biotechnol. 15, 784-788 https://doi.org/10.1038/nbt0897-784
  26. Stark, K. and Guengerich, F. P. (2007). Characterization of orphan human cytochromes P450. Drug Metab. Rev. 39, 627-637 https://doi.org/10.1080/03602530701467708
  27. Zeldin, D. C. (2001). Epoxygenase pathways of arachidonic acid metabolism. J. Biol. Chem. 276, 36059-36062 https://doi.org/10.1074/jbc.R100030200

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