The Protein Journal

, Volume 29, Issue 1, pp 32–43 | Cite as

The Role of Ile476 in the Structural Stability and Substrate Binding of Human Cytochrome P450 2C8



The biological function and stability of a cytochrome P450 (CYP) mainly depend on the subtle properties of the residues in the active site cavity, which are generally more divergent among proteins than other parts of the protein. As the most unique member of human CYP2C family, CYP2C8 has an isoleucine (Ile) 476 instead of phenylalanine (Phe) in substrate recognizing site 6 (SRS6). However, the role of Ile476 of CYP2C8 is still unknown. Therefore, six site-directed mutants of CYP2C8 were constructed to better define this. By UV–visible and circular dichroism spectroscopy studies, we studied for the first time the structural stability and all-trans-retinoic acid binding capability of the CYP2C8 variants. We found that the ferric CYP2C8 went through three states during thermal unfolding. Combined with substrate binding studies, our data revealed that residue 476 was involved in contact with substrate and was important for maintaining the thermal stability of CYP2C8.


Cytochrome P450 CYP2C8 Structural stability Unfolding Substrate binding 



Cytochrome P450


Polymerase chain reaction


Substrate recognizing site




Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Circular dichroism


The apparent substrate dissociation constant


The extrapolated maximum spectral change


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry



We thank Prof. Eric F. Johnson (The Scripps Research Institute, California, USA) for the generous gift of the pCWOri+ plasmid, encoding cytochrome P450 2C8dH. This project was supported by the National Science Foundation of China [Approving No. 20571018].


  1. 1.
    Auclair K, Moenne-Loccoz P, Ortiz de Montellano PR (2001) J Am Chem Soc 123:4877–4885CrossRefGoogle Scholar
  2. 2.
    Berry EA, Trumpower BL (1987) Anal Biochem 161:1–15CrossRefGoogle Scholar
  3. 3.
    Cavaco I, Stromberg-Norklit J, Kaneko A, Msellem MI, Dahoma M, Ribeiro VL, Bjorkman A, Gil JP (2005) Eur J Clin Pharmacol 61:15–18CrossRefGoogle Scholar
  4. 4.
    Chen CD, Doray B, Kemper B (1997) J Biol Chem 272:22891–22897CrossRefGoogle Scholar
  5. 5.
    Dai D, Zeldin DC, Blaisdell JA, Chanas B, Coulter SJ, Ghanayem BI, Goldstein JA (2001) Pharmacogenetics 11:597–607CrossRefGoogle Scholar
  6. 6.
    De Groot MJ, Alex AA, Jones BC (2002) J Med Chem 45:1983–1993CrossRefGoogle Scholar
  7. 7.
    Denisov IG, Makris TM, Sligar SG, Schlichting I (2005) Chem Rev 105:2253–2277CrossRefGoogle Scholar
  8. 8.
    Dickmann LJ, Locuson CW, Jones JP, Rettie AE (2004) Mol Pharmacol 65:842–850CrossRefGoogle Scholar
  9. 9.
    Gotoh O (1992) J Biol Chem 267:83–90Google Scholar
  10. 10.
    Haugen DA, Coon MJ (1976) J Biol Chem 251:7929–7939Google Scholar
  11. 11.
    Hichiya H, Tanaka-Kagawa T, Soyama A, Jinno H, Koyano S, Katori N, Matsushima E, Uchiyama S, Tokunaga H, Kimura H, Minami N, Katoh M, Sugai K, Goto Y, Tamura T, Yamamoto N, Ohe Y, Kunitoh H, Nokihara H, Yoshida T, Minami H, Saijo N, Ando M, Ozawa S, Saito Y, Sawada J (2005) Drug Metab Dispos 33:630–636CrossRefGoogle Scholar
  12. 12.
    Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Gene 77:51–59CrossRefGoogle Scholar
  13. 13.
    Jefcoate CR (1978) Methods Enzymol 52:258–279CrossRefGoogle Scholar
  14. 14.
    Johnson EF, Stout CD (2005) Biochem Biophys Res Commun 338:331–336CrossRefGoogle Scholar
  15. 15.
    Jung F, Griffin KJ, Song W, Richardson TH, Yang M, Johnson EF (1998) Biochemistry 37:16270–16279CrossRefGoogle Scholar
  16. 16.
    Kelly SM, Jess TJ, Price NC (2005) Biochim Biophys Acta 1751:119–139Google Scholar
  17. 17.
    Kerdpin O, Elliot DJ, Boye SL, Birkett DJ, Yoovathaworn K, Miners JO (2004) Biochemistry 43:7834–7842CrossRefGoogle Scholar
  18. 18.
    King LM, Gainer JV, David GL, Dai D, Goldstein JA, Brown NJ, Zeldin DC (2005) Pharmacogenet Genomics 15:7–13CrossRefGoogle Scholar
  19. 19.
    Klose TS, Blaisdell JA, Goldstein JA (1999) J Biochem Mol Toxicol 13:289–295CrossRefGoogle Scholar
  20. 20.
    Koo LS, Immoos CE, Cohen MS, Farmer PJ, Ortiz de Montellano PR (2002) J Am Chem Soc 124:5684–5691CrossRefGoogle Scholar
  21. 21.
    Koo LS, Tschirret-Guth RA, Straub WE, Moenne-Loccoz P, Loehr TM, Ortiz de Montellano PR (2000) J Biol Chem 275:14112–14123CrossRefGoogle Scholar
  22. 22.
    Kumar S, Sun L, Liu H, Muralidhara BK, Halpert JR (2006) Protein Eng Des Sel 19:547–554CrossRefGoogle Scholar
  23. 23.
    Kuwajima K (1989) Proteins 6:87–103CrossRefGoogle Scholar
  24. 24.
    Manna SK, Mazumdar S (2006) Biochemistry 45:12715–12722CrossRefGoogle Scholar
  25. 25.
    Martinis SA, Blanke SR, Hager LP, Sligar SG, Hoa GH, Rux JJ, Dawson JH (1996) Biochemistry 35:14530–14536CrossRefGoogle Scholar
  26. 26.
    McLean MA, Maves SA, Weiss KE, Krepich S, Sligar SG (1998) Biochem Biophys Res Commun 252:166–172CrossRefGoogle Scholar
  27. 27.
    Melet A, Assrir N, Jean P, Pilar Lopez-Garcia M, Marques-Soares C, Jaouen M, Dansette PM, Sari MA, Mansuy D (2003) Arch Biochem Biophys 409:80–91CrossRefGoogle Scholar
  28. 28.
    Melet A, Marques-Soares C, Schoch GA, Macherey AC, Jaouen M, Dansette PM, Sari MA, Johnson EF, Mansuy D (2004) Biochemistry 43:15379–15392CrossRefGoogle Scholar
  29. 29.
    Mouro C, Jung C, Bondon A, Simonneaux G (1997) Biochemistry 36:8125–8134CrossRefGoogle Scholar
  30. 30.
    Murray GI (2000) J Pathol 192:419–426CrossRefGoogle Scholar
  31. 31.
    Murugan R, Mazumdar S (2004) J Biol Inorg Chem 9:477–488CrossRefGoogle Scholar
  32. 32.
    Omura T, Sato R (1964) J Biol Chem 239:2370–2378Google Scholar
  33. 33.
    Omura T, Sato R (1964) J Biol Chem 239:2379–2385Google Scholar
  34. 34.
    Ortiz de Montellano PR (1995) Cytochrome P450: structure, mechanism and biochemistry. Plenum Press, New York, pp 473–535Google Scholar
  35. 35.
    Pace CN (1986) Methods Enzymol 131:266–280CrossRefGoogle Scholar
  36. 36.
    Pontius J, Richelle J, Wodak SJ (1996) J Mol Biol 264:121–136CrossRefGoogle Scholar
  37. 37.
    Poulos TL, Finzel BC, Howard AJ (1986) Biochemistry 25:5314–5322CrossRefGoogle Scholar
  38. 38.
    Qian W, Sun YL, Wang YH, Zhuang JH, Xie Y, Huang ZX (1998) Biochemistry 37:14137–14150CrossRefGoogle Scholar
  39. 39.
    Ridderström M, Zamora I, Fjellström O, Andersson TB (2001) J Med Chem 44:4072–4081CrossRefGoogle Scholar
  40. 40.
    Schoch GA, Yano JK, Sansen S, Dansette PM, Stout CD, Johnson EF (2008) J Biol Chem 283:17227–17237CrossRefGoogle Scholar
  41. 41.
    Schoch GA, Yano JK, Wester MR, Griffin KJ, Stout CD, Johnson EF (2004) J Biol Chem 279:9497–9503CrossRefGoogle Scholar
  42. 42.
    Snow CD, Nguyen H, Pande VS, Gruebele M (2002) Nature 420:102–106CrossRefGoogle Scholar
  43. 43.
    Totah RA, Rettie AE (2005) Clin Pharmacol Ther 77:341–352CrossRefGoogle Scholar
  44. 44.
    Wells AV, Li PS, Champion PM, Martinis SA, Sligar SG (1992) Biochemistry 31:4384–4393CrossRefGoogle Scholar
  45. 45.
    Wester MR, Stout CD, Johnson EF (2002) Methods Enzymol 357:73–79CrossRefGoogle Scholar
  46. 46.
    Wester MR, Yano JK, Schoch GA, Yang C, Griffin KJ, Stout CD, Johnson EF (2004) J Biol Chem 279:35630–35637CrossRefGoogle Scholar
  47. 47.
    Williams PA, Cosme J, Ward A, Angove HC, Matak Vinkovic D, Jhoti H (2003) Nature 424:464–468CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Chemical Biology Lab, Department of ChemistryFudan UniversityShanghaiChina

Personalised recommendations