Skip to main content
SpringerLink
Log in

Protein engineering of cytochrome P450cam

  • Chapter
  • First Online:
Metal Sites in Proteins and Models

Part of the book series: Structure and Bonding ((4143,volume 88))

Abstract

The cytochrome P450 superfamily of heme monooxygenases catalyse many reactions involved in the biosynthesis and degradation of endogenous compounds and in the oxidative metabolism of xenobiotics. These enzymes all share the same overall catalytic mechanism, and their varied substrate specificities arise from differences in their active site topologies, suggesting that it should be possible to engineer P450 enzymes for the oxidation of unnatural substrates. A review of the active site protein engineering experiments on cytochrome P450cam reported to date is presented here. Cytochrome P450cam is a stable, soluble enzyme isolated from Pseudomonas Putida, and is an excellent system for rational protein engineering. The current understanding of the details of the P450cam catalytic mechanism, including the uncoupling pathways, is first discussed. The factors controlling substrate access, binding and turnover activity, and the coupling efficiency have been investigated by site-directed mutagenesis. The results of this work have enabled the rational redesign of P450cam to broaden the substrate range, to improve the activity and coupling efficiency towards unnatural substrates, and to engineer the regioselectivity of substrate oxidation. The construction of a “self-sufficient” fusion protein containing all three proteins of the P450cam system brings one step nearer the in vitro and in vivo biotechnological applications of P450cam.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Similar content being viewed by others

References

  1. Sligar SG, Murray RI (1986) In: Ortiz de Montellano PR (ed) Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York, pp 429–503

    Google Scholar 

  2. Peterson JA, Graham-Lorence SE (1995) In: Ortiz de Montellano PR (ed) Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd edn, Plenum Press, New York, pp 151–182

    Google Scholar 

  3. Munro AW, Lindsay JG (1996) Mol Microbiol. 20:1115

    PubMed  CAS  Google Scholar 

  4. Muller HG, Schunck WH, Kargel E (1991) In: Rein H, Ruckpaul K (eds) Frontiers of Biotransformation, Taylor and Francis, London; 1991, vol. 4

    Google Scholar 

  5. Durst F (1991) In: Rein H, Ruckpaul K (eds) Frontiers of Biotransformation, Taylor and Francis, London; 1991, vol 4

    Google Scholar 

  6. Holtum JAM, Powles SB (ed) (1994) Herbicide Resistance in Plants: Biology and Biochemistry, Lewis Publishes, London

    Google Scholar 

  7. Guengerich FP (1995) In: Ortiz de Montellano PR (ed) Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd edn, Plenum Press, New York, pp 473–536

    Google Scholar 

  8. Ortiz de Montellano PR (ed) (1986) Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York

    Google Scholar 

  9. Ortiz de Montellano PR (ed) (1995) Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd edn Plenum press, New York

    Google Scholar 

  10. Cardini G, Jurtshuk P (1970) J Biol Chem 245:2789

    PubMed  CAS  Google Scholar 

  11. Asperger O, Muller R, Kleber HP (1983) Acta Biotechnol 3:319

    CAS  Google Scholar 

  12. Honeck H, Schunck W-H, Reige P, Muller H-G (1982) Biochem Biophys Res Commun 106:1318

    PubMed  CAS  Google Scholar 

  13. Guengerich FP, MacDonald TL (1984) Acc Chem Res 17:9

    CAS  Google Scholar 

  14. Guengerich FP (1991) J Biol Chem 266:10019

    PubMed  CAS  Google Scholar 

  15. Guengerich FP (1990) CRC Crit Rev Biochem 25:97

    CAS  Google Scholar 

  16. Shimada T, Martin MV, Pruess-Schwartz D, Marnett LJ, Guengerich FP (1989) Cancer Res 49:6304

    PubMed  CAS  Google Scholar 

  17. Yun C-H, Shimada T, Guengerich FP (1992) Cancer Res 52:868

    Google Scholar 

  18. McManus ME, Burgess WM, Veronese ME, Huggett A, Quattrochi LC, Tukey RH (1990) Cancer Res 50:367

    Google Scholar 

  19. Clark BJ, Waterman MR (1991) Methods Enzymol 206:00

    CAS  Google Scholar 

  20. Gonzalez FJ, Kimura S, Tamura S, Gelboin HV (1991) Methods Enzymol 206:3

    Google Scholar 

  21. Guengerich FP, Brian WR, Sari M-A, Ross J-T (1991) Methods Enzymol 206:30

    Google Scholar 

  22. Porter TD, Larson JR (1991) Methods Enzymol 206:08

    Google Scholar 

  23. Porter TD, Coon MJ (1991) J. Biol Chem 266:3469

    Google Scholar 

  24. Coon MJ, Ding X, Pernecky SJ, Vaz AND (1992) FASEB J 6:69

    Google Scholar 

  25. Katagiri M, Ganguli BN, Gunsalus IC (1968) J Biol Chem 243:543

    Google Scholar 

  26. Gunsalus IC, Wagner GC (1978) Methods Enzymol 52:66

    Google Scholar 

  27. Mueller EJ, Loida PJ, Sligar SG (1995) Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd edn. Plenum press, New York, pp 83

    Google Scholar 

  28. Peterson JA, Lu JY (1991) Methods Enzymol 206:12

    Google Scholar 

  29. Narhi LO, Fulco AJ (1987) J Biol Chem 262:683

    Google Scholar 

  30. Schaiffe A, Hutchinson CR (1987) Biochemistry 26:204

    Google Scholar 

  31. Guengerich FP (1992) Life Sci 50:471

    Google Scholar 

  32. Lindberg RLP, Negishi M (1989) Nature 339:32

    Google Scholar 

  33. Iwasaki M, Darden TA, Parker CE, Tomer KB, Pedersen LG, Negishi M (1994) J Biol Chem 269:079

    Google Scholar 

  34. Yu C-A, Gunsalus IC (1974) J Biol Chem 249:4

    Google Scholar 

  35. Unger BP, Gunsalus IC, Sligar SG (1986) J Biol Chem 261:158

    Google Scholar 

  36. Peterson J-A, Lorence MC, Amarneh B (1990) J Biol Chem 265:066

    Google Scholar 

  37. Yasukochi T, Okada O, Hara T, Sagara Y, Sekimizu K, Horiuchi T (1994) Biochim Biophys Acta 1204:4

    Google Scholar 

  38. Poulos TL, Finzel BC, Gunsalus IC, Wagner GC, Kraut J (1985) J Biol Chem 260:6122

    Google Scholar 

  39. Poulos TL, Finzel BC, Howard AJ (1987) J Mol Biol 195:87

    Google Scholar 

  40. Raag R, Poulos TL (1991) Biochemistry 30:674

    Google Scholar 

  41. Poulos TL, Howard AJ (1987) Biochemistry 26:165

    Google Scholar 

  42. Raag R, Li H, Jones BC, Poulos TL (1993) Biochemistry 32:571

    Google Scholar 

  43. Poulos TL, Finzel BC, Howard AJ (1986) Biochemistry 25:314

    Google Scholar 

  44. Sligar SG, Gunsalus IC (1976) Proc Natl Acad Sci USA 73:078

    Google Scholar 

  45. Sligar SG (1976) Biochemistry 15:399

    Google Scholar 

  46. Fisher MT, Sligar SG (1985) J Am Chem Soc 107:5018

    CAS  Google Scholar 

  47. Harris D, Loew GH (1993) J Am Chem Soc 115:775

    Google Scholar 

  48. Hintz MJ, Peterson JA (1981) J Biol Chem 256:721

    Google Scholar 

  49. Hintz MJ, Mock DM, Peterson LL, Tuttle K, Peterson JA (1982) J Biol Chem 257:4324

    Google Scholar 

  50. Stayton PS, Fisher MT, Sligar SG (1989) J Biol Chem 263:13544

    Google Scholar 

  51. Stayton PS, Poulos TL, Sligar SG (1989) Biochemistry 28:8201

    PubMed  CAS  Google Scholar 

  52. Stayton PS, Sligar SG (1990) Biochemistry 29:7381

    PubMed  CAS  Google Scholar 

  53. Geren L, Tuls J, O'Brien P, Millett F, Peterson JA (1986) J Biol Chem 261:15491

    PubMed  CAS  Google Scholar 

  54. Koga H, Sagara Y, Yaoi T, Tsujimura M, Nakamura K, Sekimizu K, Makino R, Shimada H, Ishimura Y, Yura K, Go M, Ikeguchi M, Horiuchi T (1993) FEBS Lett 331:109

    PubMed  CAS  Google Scholar 

  55. Davis MD, Sligar SG (1994) Biochemistry 31:11383

    Google Scholar 

  56. Kazlauskaite J, Westlake ACG, Wong L-L, Hill HAO (1996) J Chem Soc, Chem Commun 2189

    Google Scholar 

  57. Sligar SG, Lipscomb JD, Debrunner PG, Gunsalus IC (1974) Biochem Biophys Res Commun 61:290

    PubMed  CAS  Google Scholar 

  58. Peterson JA, Ishimura Y, Griffin BW (1972) Arch Biochem Biophys 149:197

    PubMed  CAS  Google Scholar 

  59. Groves JT, McClusky GA, White RE, Coon MJ (1978) Biochem Biophys Res Commun 81:154

    PubMed  CAS  Google Scholar 

  60. Groves JT, McClusky GA (1976) J Am Chem Soc 98:859

    CAS  Google Scholar 

  61. Atkins WM, Sligar SG (1988) Biochemistry 27:1610

    PubMed  CAS  Google Scholar 

  62. Kadkhodayan S, Coulter ED, Maryniak DM, Bryson TA, Dawson JH (1995) J Biol Chem 270:28042

    PubMed  CAS  Google Scholar 

  63. Gelb MH, Heimbrook DC, Malkonen P, Sligar SG (1982) Biochemistry 21:370

    PubMed  CAS  Google Scholar 

  64. White PW (1990) Bioorg Chem 18:440

    CAS  Google Scholar 

  65. Gunter MJ, Turner P (1991) Coord Chem Rev 108:115

    CAS  Google Scholar 

  66. Mansuy D, Battioni P (1989) Alkane Functionalization by cytochromes P450 and by model systems using O2 or H2O2. In: Hill CL (ed) Activation and Functionalization of Alkanes. Wiley, New York, p 195

    Google Scholar 

  67. Pratt J, Ridd TI, King LJ (1995) J Chem Soc, Chem Commun 2297

    Google Scholar 

  68. Lee-Robichaud P, Shyadehi AZ, Wright JN, Akhtar ME, Akhtar M (1995) Biochemistry 34:10104

    Google Scholar 

  69. Roberts ES, Vaz AND, Coon MJ (1991) Proc Natl Acad USA 88:8963

    CAS  Google Scholar 

  70. Gould P, Gelb MH, Sligar SG (1981) J Biol Chem 256:6686

    PubMed  CAS  Google Scholar 

  71. Gelb MH, Heimbrook DC, Mälkönen, P, Sligar SG (1982) Biochemistry 21:370

    PubMed  CAS  Google Scholar 

  72. Filipovic D, Paulsen MD, Loida PJ, Sligar SG, Ornstein RL (1992) Biochem Biophys Res Commun 189:488

    PubMed  CAS  Google Scholar 

  73. Ortiz de Montellano PR, Fruetel JA, Collins JR, Camper DL, Loew GH (1991) J Am Chem Soc 113:3195

    CAS  Google Scholar 

  74. Atkins WM, Sligar SG (1987) J Am Chem Soc 109:3754

    CAS  Google Scholar 

  75. Gorsky LD, Koop DR, Coon MJ (1984) J Biol Chem 259:6812

    PubMed  CAS  Google Scholar 

  76. Kuthan H, Ullrich V (1982) Eur J Biochem 126:583

    PubMed  CAS  Google Scholar 

  77. Loida PJ, Sligar SG (1993) Biochemistry 32:11530

    PubMed  CAS  Google Scholar 

  78. Haseman CA, Ravichandran KG, Peterson JA, Deisenhofer J (1994) J Mol Biol 236:1169

    Google Scholar 

  79. Cupp-Vickery J, Poulos TL (1995) Nat Struct Biol 2:144

    PubMed  CAS  Google Scholar 

  80. Ravichandran KG, Boddupalli SS, Hasemann CA, Peterson JA, Deisenhofer J (1993) Science 261:731

    PubMed  CAS  Google Scholar 

  81. Poulos TL, Cupp-Vickery J, Li H (1995) In: Ortiz de Montellano PR (ed) Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York, pp 125

    Google Scholar 

  82. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J (1995) Structure 3:41

    PubMed  CAS  Google Scholar 

  83. Nicholls A, Sharp K, Honig B (1991) Prot Struct Funct Gen 11:281

    CAS  Google Scholar 

  84. Kraulis PJ (1991) J Appl Cryst 24:946

    Google Scholar 

  85. Bacon DJ, Anderson WF (1988) J Molec Graphics 6:219

    Google Scholar 

  86. Merritt EA, Murphy MEP (1994) Acta Cryst D50:869

    CAS  Google Scholar 

  87. Griffin BW, Peterson JA (1972) Biochemistry 11:4740

    PubMed  CAS  Google Scholar 

  88. Raag R, Poulos TL (1989) Biochemistry 28:7586

    PubMed  CAS  Google Scholar 

  89. Laughton CA, Neidle S, Zvelebil MJJM, Sternberg MJE (1990) Biochem Biophys Res Commun 171:1160

    PubMed  CAS  Google Scholar 

  90. Koymans L, Vermeulen N, Baarslag A, Denkelder G (1993) J Comput Aided Mol Des 281

    Google Scholar 

  91. Boscott PE, Grant GH (1994) J Mol Graph 12:185

    PubMed  CAS  Google Scholar 

  92. Lewis DFV, Ioannides C, Parke DV (1994), Toxicol. Letts 71:235

    CAS  Google Scholar 

  93. Degroot MJ, Vermeulen N, Kramer JD, Vanacker F, Denkelder G (1996) Chem Res Toxicol 1079

    Google Scholar 

  94. Graham-Lorence S, Amarneh B, White RE, Peterson JA, Simpson ER (1995) Protein Science, vol 1065

    Google Scholar 

  95. Koymans LMH, Moereels H, Vanden-Bossche H (1995) J Steroid Biochem Mol Biol 53:191

    PubMed  CAS  Google Scholar 

  96. Szklarz GD, He YA, Halpert JR (1995) Biochemistry 34:14312

    PubMed  CAS  Google Scholar 

  97. Braatz JA, Bass MB, Ornstein RL (1994) J. Comput. Aided Mol Des 8:607

    PubMed  CAS  Google Scholar 

  98. Szklarz GD, Ornstein RL, Halpert JR, (1994) J Biomol Struct Dyn 12:61

    CAS  Google Scholar 

  99. Lin D, Zhang LH, Chiao E, Miller WL (1994) Mol Endocrinol 8:392

    PubMed  CAS  Google Scholar 

  100. De Voss JJ, Ortiz de Montellano PR (1995) J Am Chem Soc 117:4185

    Google Scholar 

  101. Sligar SG, Filipovic D, Stayton PS (1991) Methods Enzymol 206:31

    PubMed  CAS  Google Scholar 

  102. Paulsen MD, Bass MB, Ornstein RL (1991) J Biomol Struct Dyn 9:187

    PubMed  CAS  Google Scholar 

  103. Swanson BA, Dutton DR, Lunetta JM, Yang CS, Ortiz de Montellano PR (1991) J Biol Chem 266:19258

    PubMed  CAS  Google Scholar 

  104. Tuck SF, Graham-Lorence S, Peterson JA, Ortiz de Montellano PR (1993) J Biol Chem 268:269 and references therein

    PubMed  CAS  Google Scholar 

  105. Atkins WM, Sligar SG (1988) J Biol Chem 263:18842

    PubMed  CAS  Google Scholar 

  106. Fisher MT, Sligar SG (1987) Biochemistry 26:4797

    PubMed  CAS  Google Scholar 

  107. Atkins WM, Sligar SG (1989) J Am Chem Soc 111:2716

    Google Scholar 

  108. Martinis SA, Atkins WM, Stayton PS, Sligar SG (1989) J Am Chem Soc 111:9252

    CAS  Google Scholar 

  109. Imai M, Shimada H, Watanabe Y, Matsushima-Hibaya Y, Makino R, Koga H, Horiuchi T, Ishimura Y (1989) Proc Natl Acad Sci USA 86:7823

    PubMed  CAS  Google Scholar 

  110. Kimata Y, Shimada T, Ishimura Y (1995) Biochem. Biophys Res Commun 208:96

    PubMed  CAS  Google Scholar 

  111. Raag R, Martinis SA, Sligar SG, Poulos TL (1991) Biochemistry 30:11420

    PubMed  CAS  Google Scholar 

  112. Aikens J, Sligar SG (1994) J Am Chem Soc 116:1143

    CAS  Google Scholar 

  113. Gerber NC, Sligar SG (1992) J Am Chem Soc 114:8742

    CAS  Google Scholar 

  114. Gerber NC, Sligar SG (1994) J Biol Chem 269:4260

    PubMed  CAS  Google Scholar 

  115. Shimada H, Makino R, Unno M, Horiuchi T, Ishimura Y (1993) In: Lechner MC (ed) Cytochrome P450 Biochemistry, Biophysics and Molecular Biology, John Libbey, pp 299

    Google Scholar 

  116. Loida PJ, Sligar SG (1993) Protein Eng 2:207

    Google Scholar 

  117. Fruetel JA, Collins JR, Camper DL, Loew GH, Ortiz de Montellano PR (1992) J Am Chem Soc 114:6987

    CAS  Google Scholar 

  118. Watanabe Y, Ishimura Y (1989) J Am Chem Soc 111:410

    CAS  Google Scholar 

  119. Jones JP, Trager WF, Carlson TJ (1993) J Am Chem Soc 115:381

    CAS  Google Scholar 

  120. Grayson DA, Tewari YB, Mayhew MP, Vilker VL, Goldberg RN (1996) Arch Biochem Biophys 332:239

    PubMed  CAS  Google Scholar 

  121. Castro CE, Wade RS, Belser NO (1985) Biochemistry 24:204

    PubMed  CAS  Google Scholar 

  122. Li S, Wackett LP (1993) Biochemistry 32:9355

    PubMed  CAS  Google Scholar 

  123. Lefever MR, Wackett LP (1994) Biochem Biophys Res Commun 201:373

    PubMed  CAS  Google Scholar 

  124. Stevenson JA, Westlake ACG, Whittock C, Wong L-L (1996) J Am Chem Soc, 118:12846

    CAS  Google Scholar 

  125. Fowler SM, England PA, Westlake ACG, Rouch DA, Nickerson DP, Blunt C, Braybrook D, West S, Wong L-L, Flitsch SL (1994) J Chem Soc, Chem Commun 2761

    Google Scholar 

  126. Bell SG, Rouch DA, Wong LL, J Mol Catal, in press

    Google Scholar 

  127. England PA, Rouch DA, Westlake ACG, Bell SG, Nickerson DP, Webberley M, Flitsch SL, Wong L-L (1996), J Chem Soc, Chem Commun 357

    Google Scholar 

  128. Jones NE, England PA, Rouch DA, Wong L-L (1996) J Chem Soc Chem Commun 2413

    Google Scholar 

  129. Murakami H, Yabusaki Y, Sakaki T, Shibata M, Ohkawa H (1987) DNA Cell Biol 6:189

    CAS  Google Scholar 

  130. Shet MS, Faulkner KM, Holmans PL, Fisher CW, Estabrook RW (1995) Arch Biochem Biophys 318:314

    PubMed  CAS  Google Scholar 

  131. Harikrishner JA, Black SM, Szklarz GD, Miller WL (1993) DNA Cell Biol 12:371

    Google Scholar 

  132. Sibbesen O, De Voss JJ, Ortiz de Montellano PR (1996) J Biol Chem 271:22462

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Inorganic Chemistry Laboratory, South Parks Road, OX1 3QR, Oxford, UK

    Luet-Lok Wong, Andrew C. G. Westlake & Darren P. Nickerson

Authors
  1. Luet-Lok Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew C. G. Westlake
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Darren P. Nickerson
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

H. A. O. Hill P. J. Sadler A. J. Thomson

Rights and permissions

Reprints and permissions

Copyright information

© 1997 Springer Verlag

About this chapter

Cite this chapter

Wong, LL., Westlake, A.C.G., Nickerson, D.P. (1997). Protein engineering of cytochrome P450cam . In: Hill, H.A.O., Sadler, P.J., Thomson, A.J. (eds) Metal Sites in Proteins and Models. Structure and Bonding, vol 88. Springer, Berlin, Heidelberg. https://doi.org/10.1007/3-540-62870-3_6

Download citation

  • DOI: https://doi.org/10.1007/3-540-62870-3_6

  • Published:

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-540-62870-5

  • Online ISBN: 978-3-540-69035-1

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation