JBIC Journal of Biological Inorganic Chemistry

, Volume 10, Issue 8, pp 842–853

Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego

  • Michael Fairhead
  • Silva Giannini
  • Elizabeth M. J. Gillam
  • Gianfranco Gilardi
Original Article

Abstract

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1–BMR, contains the N-terminally modified residues 22–493 of the human P450 2E1 fused at the C-terminus to residues 473–1049 of the P450 BM3 reductase (BMR). The P450 2E1–BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84±0.09 mM and kcat of 2.98±0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65±0.08 mM and kcat of 0.95±0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

Keywords

Structure–function relationship Protein engineering Cytochrome Nanotechnolgy Electron transfer 

Abbreviations

δ-ALA

δ-Aminolevulenic acid

BMR

Cytochrome P450 reductase domain of P450 BM3

CPR

Cytochrome P450 NADPH-dependent oxidoreductase

DEAE

(Diethylamino)ethyl

DTT

Dithiothreitol

HPLC

High-performance liquid chromatography

IPTG

Isopropyl-β-D-thiogalactopyranoside

P450 BM3

Bacterial cytochrome P450 from Bacillus megaterium

P450 BMP

Haem domain of P450 BM3

P450 2E1

Human cytochrome P450 2E1

SRS

Substrate recognition site

References

  1. 1.
    Poulos TL (1995) Cytochrome P450. Curr Opin Struct Biol 5:767–774CrossRefPubMedGoogle Scholar
  2. 2.
    Nelson DR (1999) Cytochrome P450 and the individuality of species. Arch Biochem Biophys 369:1–15CrossRefPubMedGoogle Scholar
  3. 3.
    Hasler JA, Estabrook RW, Murray M, Pikuleva I, Waterman M, Capdevila J, Holla V, Helvig C, Falck JR, Farrell G, Kaminsky LS, Spivack SD, Boitier E, Beaune P (1999) Human cytochrome P450. Mol Aspects Med 20:1–137CrossRefGoogle Scholar
  4. 4.
    Guengerich FP (2001) Common and uncommon cytochrome P450 reactions related to metabolism and toxicity. Chem Res Toxicol 14:611–650PubMedCrossRefGoogle Scholar
  5. 5.
    Nelson DR, Strobel HW (1988) On the membrane topology of vertebrate cytochrome P450 proteins. J Biol Chem 263:6038–6050PubMedGoogle Scholar
  6. 6.
    Guengerich FP, Kim DH, Iwasaki M (1991) Role of human cytochrome P450 2E1 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 4:168–179CrossRefPubMedGoogle Scholar
  7. 7.
    Lieber CS (1997) Cytochrome P-4502E1: its physiological and pathological role. Physiol Rev 77:517–538PubMedGoogle Scholar
  8. 8.
    Tanaka E, Terada M, Misawa S (2000) Cytochrome P450 2E1: its clinical and toxicological role. J Clin Pharm Ther 25:165–175CrossRefPubMedGoogle Scholar
  9. 9.
    Chen W, Koenigs LL, Thompson SJ, Peter RM, Rettie AE, Trager WF, Nelson SD (1998) Oxidation of acetaminophen to its toxic quinone imine and nontoxic catechol metabolites by baculovirus-expressed and purified human cytochromes P450 2E1 and 2A6. Chem Res Toxicol 11:295–301CrossRefPubMedGoogle Scholar
  10. 10.
    Peter R, Bocker R, Beaune PH, Iwasaki M, Guengerich FP, Yang CS (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1. Chem Res Toxicol 3:566–573CrossRefPubMedGoogle Scholar
  11. 11.
    Koop DR (1986) Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P-450 isozyme 3a. Mol Pharmacol 29:399–404PubMedGoogle Scholar
  12. 12.
    Fantuzzi A, Fairhead M, Gilardi G (2004) Direct electrochemistry of immobilized human cytochrome P450 2E1. J Am Chem Soc 126:5040–5041CrossRefPubMedGoogle Scholar
  13. 13.
    Narhi LO, Fulco AJ (1986) Characterization of a catalytically self-sufficient 119,000-Dalton cytochrome P-450 monooxygenase induced by barbituates in Bacillus megaterium. J Biol Chem 261:7160–7169PubMedGoogle Scholar
  14. 14.
    Narhi LO, Fulco AJ (1987) Identification and characterisation of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbituates in Bacillus megaterium. J Biol Chem 262:6683–6690PubMedGoogle Scholar
  15. 15.
    Lewis DF, Hlavica P (2000) Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects. Biochim Biophys Acta 1460:353–374PubMedCrossRefGoogle Scholar
  16. 16.
    Boddupalli SS, Estabrook RW, Peterson JA (1990) Fatty acid monooxygenation by cytochrome P-450BM-3. J Biol Chem 265:4233–4239PubMedGoogle Scholar
  17. 17.
    Fang C, Koyabashi Y, Halpert JR (1997) Stoichometry of 7-ethoxycoumarin metabolism by cytochrome P450 2B1 wild-type and five active-site mutants. FEBS Lett 416:77–80CrossRefPubMedGoogle Scholar
  18. 18.
    Mayuzumi H, Sambongi C, Hiroya K, Shimizu T, Tateishi T, Hatano M (1993) Effect of mutations of ionic amino acids of cytochrome P450 1A2 on catalytic activities toward 7-ethoxycoumarin and methanol. Biochemistry 32:5622–5628CrossRefPubMedGoogle Scholar
  19. 19.
    Perret A, Pompon D (1998) Electron shuttle between membrane-bound cytochrome P450 3A4 and b5 rules uncoupling mechanisms. Biochemistry 37:11412–11424CrossRefPubMedGoogle Scholar
  20. 20.
    Yun CH, Miller GP, Guengerich FP (2000) Rate-determining steps in phenacetin oxidations by human cytochrome P450 1A2 and selected mutants. Biochemistry 39:11319–11329PubMedCrossRefGoogle Scholar
  21. 21.
    Noble MA, Miles CS, Chapman SK, Lysek DA, Mackay AC, Reid GA, Hanzlick RP, Munro AW (1999) Roles of key active-site residues in flavocytochrome P450 BM3. Biochem J 339:371–379CrossRefPubMedGoogle Scholar
  22. 22.
    Munro AW, Leys DG, McLean KJ, Marshall KR, Ost TW, Daff S, Miles CS, Chapman SK, Lysek DA, Moser CC, Page CC, Dutton PL (2002) P450 BM3: the very model of a modern flavocytochrome. Trends Biochem Sci 27:250–257PubMedCrossRefGoogle Scholar
  23. 23.
    Giannini S, Fairhead MJ, Gilardi G (2001) Engineering a soluble, catalytically self-sufficient human P450 for nanobiotechnology. Biochem Soc Trans 29:31Google Scholar
  24. 24.
    Gilardi G, Meharenna YT, Tsotsou GE, Sadeghi SJ, Fairhead M, Giannini S (2002) Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology. Biosens Bioelectron 17:133–145CrossRefPubMedGoogle Scholar
  25. 25.
    Fisher CW, Schet MS, Caule DL, Martin-Wintrom CA, Estabrook RW (1992) High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes p450 and NADPH-p450 reductase flavoprotein. Proc Natl Acad Sci USA 89:10817–10821PubMedCrossRefGoogle Scholar
  26. 26.
    Shet MS, Fisher CW, Arlotto MP, Shakleton CHL, Holmans PL, Martin-Wixtrom CA, Saeki Y, Estabrook RW (1994) Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine 17A and rat NADPH-P450 reductase. Arch Biochem Biophys 311:402–417CrossRefPubMedGoogle Scholar
  27. 27.
    Shet MS, Fisher CW, Holmans PL, Estabrook RW (1993) Human cytochrome p450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-p450 reductase. Proc Natl Acad Sci USA 90:11748–11752PubMedCrossRefGoogle Scholar
  28. 28.
    Harlow GR, Halpert JR (1996) Mutagenesis study of Asp-290 in cytochrome p450 2B11 using a fusion protein with rat NADPH-cytochrome p450 reductase. Arch Biochem Biophys 326:85–92CrossRefPubMedGoogle Scholar
  29. 29.
    Lu P, Alterman MA, Chaurasia CS, Bambal RB, Hanzlick RP (1997) Heme-coordinating analogs of lauric acid as inhibitors of fatty acid ω-hydroxylation. Arch Biochem Biophys 337:1–7CrossRefPubMedGoogle Scholar
  30. 30.
    Gilardi G, Meharenna YT, Tsotsou GE, Sadeghi SJ, Fairhead M, Giannini S (2002) Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology. Biosens Bioelectron 17(1–2):133–145CrossRefPubMedGoogle Scholar
  31. 31.
    Sadeghi SJ, Meharenna YT, Fantuzzi A, Valetti F, Gilardi G (2000) Engineering artificial redox chains by Molecular Lego. Faraday Discuss 116:135–153CrossRefPubMedGoogle Scholar
  32. 32.
    Gillam EM, Guo Z, Guengerich FP (1994) Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties. Arch Biochem Biophys 312:59–66CrossRefPubMedGoogle Scholar
  33. 33.
    Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver micrososmes. J Biol Chem 239:2370–2385PubMedGoogle Scholar
  34. 34.
    Ozols J, Strittmatter P (1964) The interaction of porphyrins and metalloporphyrins with apocytochrome b5. J Biol Chem 239:1018–1023PubMedGoogle Scholar
  35. 35.
    Yuan R, Madani S, Wei XX, Reynolds K, Huang SM (2002) Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos 30:1311–1319CrossRefPubMedGoogle Scholar
  36. 36.
    Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723CrossRefPubMedGoogle Scholar
  37. 37.
    Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  38. 38.
    Pontius J, Richelle J, Wodak SJ (1996) Quality assessment of protein 3D structures using standard atomic volumes. J Mol Biol 264:121–136CrossRefPubMedGoogle Scholar
  39. 39.
    Vriend G (1996) WHAT IF: a molecular modeling and drug design program. J Mol Graph 8:52–56CrossRefGoogle Scholar
  40. 40.
    Valetti F, Sadeghi SJ, Meharenna YT, Leliveld SR, Gilardi G (1998) Engineering multi-domain redox proteins containing flavodoxin as bio-transformer: preparatory studies by rational design. Biosens Bioelectron 13:675–685CrossRefPubMedGoogle Scholar
  41. 41.
    Nishimoto M, Clark JE, Masters BSS (1993) Cytochrome P450 4A4: expression in Escherichia coli, purification, and characterisation of catalytic properties. Biochemistry 32:8863–8870CrossRefPubMedGoogle Scholar
  42. 42.
    Gillam EMJ, Guo Z, Martin MV, Jenkins CM, Guengerich FP (1995) Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch Biochem Biophys 319:540–550CrossRefPubMedGoogle Scholar
  43. 43.
    Kery V, Elleder D, Kraus JP (1995) δ-Aminolevulinate increase heme saturation and yield of human cystathionine β-synthase expressed in Escherichia coli. Arch Biochem Biophys 316:24–29CrossRefPubMedGoogle Scholar
  44. 44.
    Woodard SI, Dailey HA (1995) Regulation of heme biosynthesis in Escherichia coli. Arch Biochem Biophys 316:110–115CrossRefPubMedGoogle Scholar
  45. 45.
    Koop DR (1990) Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino-1,2,4-triazole. Chem Res Toxicol 3:377–383CrossRefPubMedGoogle Scholar
  46. 46.
    Porter TD (1994) Mutagenesis at a highly conserved phenylalanine in cytochrome P450 2E1 affects heme incorporation and catalytic activity. Biochemistry 33:5942–5946CrossRefPubMedGoogle Scholar
  47. 47.
    Larson JR, Coon MJ, Porter TD (1991) Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli. J Biol Chem 266:7321–7324PubMedGoogle Scholar
  48. 48.
    Dong JS, Porter TD (1996) Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli. Arch Biochem Biophys 327:254–259CrossRefPubMedGoogle Scholar
  49. 49.
    Zerilli A, Ratanasavanh D, Lucas D, Goasduff T, Dreano Y, Menard C, Picart D, Berthou F (1997) Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for p450 2E1. Chem Res Toxicol 10:1205–1212CrossRefPubMedGoogle Scholar
  50. 50.
    Chen W, Peter RM, McArdle S, Thummel KE, Sigle RO, Nelson SD (1996) Baculovirus expression and purification of human and rat cytochrome P450 2E1. Arch Biochem Biophys 335:123–130CrossRefPubMedGoogle Scholar
  51. 51.
    Umeno M, McBride OW, Yang CS, Gelboin HV, Ginzalez FJ (1988) Human ethanol-inducible P450IIEI: complete gene sequence, promoter characterization, chromosome mapping and cDNA-directed expression. Biochemistry 27:9006–9013CrossRefPubMedGoogle Scholar
  52. 52.
    Sevrioukova IF, Li H, Zhang H, Peterson JA, Poulos TL (1999) Structure of a cytochrome P450-redox partner electron-transfer complex. Proc Natl Acad Sci USA 96:1863–1868CrossRefPubMedGoogle Scholar
  53. 53.
    Gotoh O (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 267:83–90PubMedGoogle Scholar
  54. 54.
    Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE (2000) Mammalian microsomal cytochrome p450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell 5:121–131CrossRefPubMedGoogle Scholar
  55. 55.
    Gorsky LD, Koop DR, Coon MJ (1984) On the stoichiometry of the oxidase and monooxygenase reactions catalysed by liver microsomal P-450. J Biol Chem 259:6812–6817PubMedGoogle Scholar
  56. 56.
    Albano EA (1991) Role of ethanol inducible cytochrome p450 (p450IIE1) in catalyzing the free radicle activation of aliphatic alcohols. Biochem Pharmocol 41:1895–1902CrossRefGoogle Scholar
  57. 57.
    Wang MH, Patten CJ, Yang GY, Paranawithana SR, Tan Y, Yang CS (1996) Expression and coupling of human cytochrome P450 2E1 and NADPH-cytochrome P450 oxidoreductase in dual expression and co-infection systems with baculovirus in insect cells. Arch Biochem Biophys 334:380–388CrossRefPubMedGoogle Scholar
  58. 58.
    Patten CJ, Koch P (1995) Baculovirus expression of human P450 2E1 and cytochrome b5: spectral and catalytic properties and effect of b5 on the stoichiometry of P450 2E1-catalyzed reactions. Arch Biochem Biophys 317:504–513CrossRefPubMedGoogle Scholar
  59. 59.
    Bridges A, Gruenke L, Chang YT, Vasker IA, Loew G, Waskell L (1998) Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 & cytochrome P450 reductase. J Biol Chem 273:17036–17049CrossRefPubMedGoogle Scholar
  60. 60.
    Helvig C, Capdevila JH (2000) Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains. Biochemistry 39:5196–5205CrossRefPubMedGoogle Scholar
  61. 61.
    Davydov DR, Kariakin AA, Petushkova NA, Peterson JA (2000) Association of cytochromes P450 with their reductases: opposite sign of the electrostatic interactions in P450BM-3 as compared with the microsomal 2B4 system. Biochemistry 39:6489–6497CrossRefPubMedGoogle Scholar
  62. 62.
    Zhao Q, Modi S, Smith G, Paine M, McDonagh PD, Wolf CR, Tew D, Lian LY, Roberts GC, Driessen HP (1999) Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 Å resolution. Protein Sci 8:298–306PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2005

Authors and Affiliations

  • Michael Fairhead
    • 1
    • 2
  • Silva Giannini
    • 1
    • 2
  • Elizabeth M. J. Gillam
    • 3
  • Gianfranco Gilardi
    • 1
    • 2
    • 4
  1. 1.Division of Molecular Biosciences, Faculty of Life SciencesImperial College LondonLondonUK
  2. 2.NanoBiodesign Ltd.LondonUK
  3. 3.Department of Physiology & Pharmacology, School of Biomedical SciencesThe University of QueenslandBrisbaneAustralia
  4. 4.Department of Human and Animal BiologyUniversity of TurinTurinItaly

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