Advertisement

Cytochrome P450cin (CYP176A1)

  • Jeanette E. Stok
  • Kate E. Slessor
  • Anthony J. Farlow
  • David B. Hawkes
  • James J. De VossEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 851)

Abstract

Cytochrome P450cin (P450cin) (CYP176A1) is a bacterial P450 enzyme that catalyses the enantiospecific hydroxylation of 1,8-cineole to (1R)-6β-hydroxycineole when reconstituted with its natural reduction-oxidation (redox) partner cindoxin, E. coli flavodoxin reductase, and NADPH as a source of electrons. This catalytic system has become a useful tool in the study of P450s as not only can large quantities of P450cin be prepared and rates of oxidation up to 1,500 min−1 achieved, but it also displays a number of unusual characteristics. These include an asparagine residue in P450cin that has been found in place of the usual conserved threonine residue observed in most P450s. In general, this conserved threonine controls oxygen activation to create the potent ferryl (Fe(IV=O) porphyrin cation radical required for substrate oxidation. Another atypical characteristic of P450cin is that it utilises an FMN-containing redoxin (cindoxin) rather than a ferridoxin as is usually observed with other bacterial P450s (e.g. P450cam). This chapter will review what is currently known about P450cin and how this enzyme has provided a greater understanding of P450s in general.

Keywords

Cytochrome P450cin CYP176A1 Cindoxin Cindoxin reductase Ferric hydroperoxy species Ferryl porphyrin radical species 

Notes

Acknowledgements

The authors would like to acknowledge that this work was supported in part by ARC Grants DP110104455 and DP140103229.

References

  1. 1.
    Guengerich FP (2005) Human cytochrome P450 enzymes. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry, 3rd edn. Kluwer Academic/Plenum Publishers, New York, pp 377–530CrossRefGoogle Scholar
  2. 2.
    Ortiz de Montellano PR (ed) (2005) Cytochrome P450: structure, mechanism, and biochemistry. Kluwer Academic/Plenum Publishers, New YorkGoogle Scholar
  3. 3.
    Poulos TL, Finzel BC, Gunsalus IC, Wagner GC, Kraut J (1985) The 2.6-Å crystal-structure of Pseudomonas putida cytochrome P450. J Biol Chem 260:6122–6130Google Scholar
  4. 4.
    Poulos TL, Finzel BC, Howard AJ (1987) High-resolution crystal structure of cytochrome P450cam. J Mol Biol 195:687–700CrossRefPubMedGoogle Scholar
  5. 5.
    Mueller EJ, Loida PJ, Sligar SG (1995) Twenty-five years of P450cam research. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry, 2nd edn. Plenum Press, New York, pp 83–124CrossRefGoogle Scholar
  6. 6.
    Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE, Sweet BM, Ringe D, Petsko GA, Sligar SG (2000) The catalytic pathway of cytochrome P450cam at atomic resolution. Science 287:1615–1622CrossRefPubMedGoogle Scholar
  7. 7.
    Poulos T (2005) Structural biology of heme monooxygenases. Biochem Biophys Res Commun 338:337–345CrossRefPubMedGoogle Scholar
  8. 8.
    Whitehouse CJC, Bell SG, Wong LL (2012) P450BM3 (CYP102A1): connecting the dots. Chem Soc Rev 41:1218–1260CrossRefPubMedGoogle Scholar
  9. 9.
    Deprez E, Di Primo C, Hui Bon Hoa G, Douzou P (1994) Effects of monovalent cations on cytochrome P-450 camphor evidence for preferential binding of potassium. FEBS Lett 347:207–210CrossRefPubMedGoogle Scholar
  10. 10.
    Hawkes DB, Adams GW, Burlingame AL, Ortiz de Montellano PR, De Voss JJ (2002) Cytochrome P450cin (CYP176A), isolation, expression, and characterization. J Biol Chem 277:27725–27732CrossRefPubMedGoogle Scholar
  11. 11.
    Kim D, Heo Y-S, Ortiz de Montellano PR (2008) Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation. Arch Biochem Biophys 474:150–156CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Meharenna YT, Slessor KE, Cavaignac SM, Poulos TL, De Voss JJ (2008) The critical role of substrate-protein hydrogen bonding in the control of regioselective hydroxylation in P450cin. J Biol Chem 283:10804–10812CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Slessor KE, Farlow AJ, Cavaignac SM, Stok JE, De Voss JJ (2011) Oxygen activation by P450cin: protein and substrate mutagenesis. Arch Biochem Biophys 507:154–162CrossRefPubMedGoogle Scholar
  14. 14.
    Kimmich N, Das A, Sevrioukova I, Meharenna Y, Sligar SG, Poulos TL (2007) Electron transfer between cytochrome P450cin and its FMN-containing redox partner, cindoxin. J Biol Chem 282:27006–27011CrossRefPubMedGoogle Scholar
  15. 15.
    Hawkes DB, Slessor KE, Bernhardt PV, De Voss JJ (2010) Cloning, expression and purification of cindoxin, an unusual FMN-containing cytochrome P450 redox partner. ChemBioChem 11:1107–1114CrossRefPubMedGoogle Scholar
  16. 16.
    Slessor KE, Stok JE, Cavaignac SM, Hawkes DB, Ghasemi Y, De Voss JJ (2010) Cineole biodegradation: molecular cloning, expression and characterisation of (1R)-6 β-hydroxycineole dehydrogenase from Citrobacter braakii. Bioorg Chem 38:81–86CrossRefPubMedGoogle Scholar
  17. 17.
    Meharenna YT, Li H, Hawkes DB, Pearson AG, De Voss J, Poulos TL (2004) Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam. Biochemistry 43:9487–9494CrossRefPubMedGoogle Scholar
  18. 18.
    Madrona Y, Tripathi S, Li H, Poulos TL (2012) Crystal structures of substrate-free and nitrosyl cytochrome P450cin: implications for O2 activation. Biochemistry 51:6623–6631CrossRefPubMedGoogle Scholar
  19. 19.
    Ost T, Miles C, Munro A, Murdoch J, Reid G, Chapman S (2001) Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3. Biochemistry 40:13421–13429CrossRefPubMedGoogle Scholar
  20. 20.
    Paine MJI, Scrutton NS, Munro AW, Gutierrez A, Roberts GCK, Wolf CR (2005) Electron transfer partners of cytochrome P450. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry, 3rd edn. Kluwer Academic/Plenum Publishers, New York, pp 115–148CrossRefGoogle Scholar
  21. 21.
    Lipscomb JD, Sligar SG, Namtvedt MJ, Gunsalus IC (1976) Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam. J Biol Chem 251:1116–1124PubMedGoogle Scholar
  22. 22.
    Bernhardt R, Gunsalus IC (1992) Reconstitution of cytochrome P4502B4 (LM2) activity with camphor and linalool monooxygenase electron donors. Biochem Biophys Res Commun 187:310–317CrossRefPubMedGoogle Scholar
  23. 23.
    Peterson JA, Graham-Lorence SE (1995) Bacterial P450s. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry, 2nd edn. Plenum Press, New York, pp 151–180CrossRefGoogle Scholar
  24. 24.
    Ullah AJ, Murray RI, Bhattacharyya PK, Wagner GC, Gunsalus IC (1990) Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase. J Biol Chem 265:1345–1351PubMedGoogle Scholar
  25. 25.
    Hannemann F, Bichet A, Ewen K, Bernhardt R (2007) Cytochrome P450 systems—biological variations of electron transport chains. Biochim Biophys Acta 1770:330–344CrossRefPubMedGoogle Scholar
  26. 26.
    Ewen KM, Kleser M, Bernhardt R (2011) Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins. Biochim Biophys Acta 1814:111–125CrossRefPubMedGoogle Scholar
  27. 27.
    Waterman M, Jenkins C, Pikuleva I (1995) Genetically engineered bacterial cells and applications. Toxicol Lett 82:807–813CrossRefPubMedGoogle Scholar
  28. 28.
    Holden M, Mayhew M, Bunk D, Roitberg A, Vilker V (1997) Probing the interactions of putidaredoxin with redox partners in camphor P450 5-monooxygenase by mutagenesis of surface residues. J Biol Chem 272:21720–21725CrossRefPubMedGoogle Scholar
  29. 29.
    Pochapsky T, Ye X, Ratnaswamy G, Lyons T (1994) An NMR-derived model for the solution structure of oxidized putidaredoxin, a 2-Fe, 2-S ferredoxin from Pseudomonas. Biochemistry 33:6424–6432CrossRefPubMedGoogle Scholar
  30. 30.
    Pochapsky TC, Lyons TA, Kazanis S, Arakaki T, Ratnaswamy G (1996) A structure-based model for cytochrome P450cam-putidaredoxin interactions. Biochimie 78:723–733CrossRefPubMedGoogle Scholar
  31. 31.
    Madrona Y, Hollingsworth SA, Tripathi S, Fields JB, Rwigema J-CN, Tobias DJ, Poulos TL (2014) Crystal structure of cindoxin, the P450cin redox partner. Biochemistry 53:1435–1446CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Sligar S, Gunsalus I (1976) A thermodynamic model of regulation: modulation of redox equilibria in camphor monoxygenase. Proc Natl Acad Sci U S A 73:1078–1082CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Aguey-Zinsou K-F, Bernhardt PV, De Voss JJ, Slessor KE (2003) Electrochemistry of P450cin: new insights into P450 electron transfer. Chem Commun 418–419Google Scholar
  34. 34.
    Stok JE, De Voss JJ (2000) Expression, purification, and characterization of Biol: a carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis. Arch Biochem Biophys 384:351–360CrossRefPubMedGoogle Scholar
  35. 35.
    Hawkes DB (2003) Cytochrome P450cin, Ph.D. thesis, The University of Queensland, BrisbaneGoogle Scholar
  36. 36.
    Slessor KE (2007) Cytochrome P450cin: chemistry and biochemistry, Ph.D. thesis, The University of Queensland, BrisbaneGoogle Scholar
  37. 37.
    Martinis SA, Atkins WM, Stayton PS, Sligar SG (1989) A conserved residue of cytochrome P450 is involved in heme-oxygen stability and activation. J Am Chem Soc 111:9252–9253CrossRefGoogle Scholar
  38. 38.
    Andersen JF, Tatsuta K, Gunji H, Ishiyama T, Hutchinson CR (1993) Substrate specificity of 6-deoxyerythronolide B hydroxylase, a bacterial cytochrome P450 of erythromycin A biosynthesis. Biochemistry 32:1905–1913CrossRefPubMedGoogle Scholar
  39. 39.
    Xiang H, Tschirret-Guth RA, Ortiz de Montellano PR (2000) An A245T mutation conveys on cytochrome P450EryF the ability to oxidize alternative substrates. J Biol Chem 275:35999–36006CrossRefPubMedGoogle Scholar
  40. 40.
    Clark JP, Miles CS, Mowat CG, Walkinshaw MD, Reid GA, Simon NDA, Chapman SK (2006) The role of Thr268 and Phe393 in cytochrome P450BM3. J Inorg Biochem 100:1075–1090CrossRefPubMedGoogle Scholar
  41. 41.
    Atkins WM, Sligar SG (1988) The roles of active-site hydrogen-bonding in cytochrome P450cam as revealed by site-directed mutagenesis. J Biol Chem 263:18842–18849PubMedGoogle Scholar
  42. 42.
    Deprez E, Gill E, Helms V, Wade RC, Hui Bon Hoa G (2002) Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin. J Inorg Biochem 91:597–606CrossRefPubMedGoogle Scholar
  43. 43.
    Loida PJ, Sligar SG, Paulsen MD, Arnold GE, Ornstein RL (1995) Stereoselective hydroxylation of norcamphor by cytochrome P450cam−experimental verification of molecular-dynamics simulations. J Biol Chem 270:5326–5330CrossRefPubMedGoogle Scholar
  44. 44.
    Slessor KE, Hawkes DB, Farlow A, Pearson AG, Stok JE, De Voss JJ (2012) An in vivo cytochrome P450cin (CYP176A1) catalytic system for metabolite production. J Mol Catal B: Enzym 79:15–20CrossRefGoogle Scholar
  45. 45.
    Slessor KE, Stok JE, Chow S, De Voss JJ, Unpublished resultsGoogle Scholar
  46. 46.
    Gerber NC, Sligar SG (1992) Catalytic mechanism of cytochrome-P450−evidence for a distal charge relay. J Am Chem Soc 114:8742–8743CrossRefGoogle Scholar
  47. 47.
    Gerber NC, Sligar SG (1994) A role for Asp-251 in cytochrome P450cam oxygen activation. J Biol Chem 269:4260–4266PubMedGoogle Scholar
  48. 48.
    Stok JE, Yamada S, Farlow AJ, Slessor KE, De Voss JJ (2013) Cytochrome P450cin (CYP176A1) D241N: investigating the role of the conserved acid in the active site of cytochrome P450s. Biochim Biophys Acta 1834:688–696CrossRefPubMedGoogle Scholar
  49. 49.
    Davydov R, Macdonald IDG, Makris TM, Sligar SG, Hoffman BM (1999) EPR and ENDOR of catalytic intermediates in cryoreduced native and mutant oxy-cytochromes P450cam: mutation-induced changes in the proton delivery system. J Am Chem Soc 121:10654–10655CrossRefGoogle Scholar
  50. 50.
    Davydov R, Makris TM, Kofman V, Werst DE, Sligar SG, Hoffman BM (2001) Hydroxylation of camphor by-reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes. J Am Chem Soc 123:1403–1415CrossRefPubMedGoogle Scholar
  51. 51.
    Benson DE, Suslick KS, Sligar SG (1997) Reduced oxy intermediate observed in D251N cytochrome P450cam. Biochemistry 36:5104–5107CrossRefPubMedGoogle Scholar
  52. 52.
    Akhtar M, Corina D, Pratt J, Smith T (1976) Studies on removal of C-19 in estrogen biosynthesis using 18O2. J Chem Soc Chem Commun 854–856Google Scholar
  53. 53.
    Akhtar M, Njar VCO, Wright JN (1993) Mechanistic studies on aromatase and related C-C bond cleaving P450 enzymes. J Steroid Biochem 44:375–387CrossRefGoogle Scholar
  54. 54.
    Gantt SL, Denisov IG, Grinkova YV, Sligar SG (2009) The critical iron-oxygen intermediate in human aromatase. Biochem Biophys Res Commun 387:169–173CrossRefPubMedCentralPubMedGoogle Scholar
  55. 55.
    Stevenson DE, Wright JN, Akhtar M (1988) Mechanistic consideration of P450 dependent enzymic reactions−studies on estriol biosynthesis. J Chem Soc Perkin Trans 1:2043–2052CrossRefGoogle Scholar
  56. 56.
    Ortiz de Montellano PR, De Voss JJ (2005) Substrate oxidation by cytochrome P450 enzymes. In: Ortiz de Montellano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry, 3rd edn. Kluwer Academic/Plenum Publishers, New York, pp 183–245CrossRefGoogle Scholar
  57. 57.
    Carman RM, Fletcher MT (1983) Halogenated terpernoids. XX. The seven monochlorocineoles. Aust J Chem 36:1483–1493CrossRefGoogle Scholar
  58. 58.
    Macrae IC, Alberts V, Carman RM, Shaw IM (1979) Products of 1,8-cineole oxidation by a pseudomonad. Aust J Chem 32:917–922CrossRefGoogle Scholar
  59. 59.
    Williams DR, Trudgill PW, Taylor DG (1989) Metabolism of 1,8-cineole by a Rhodococcus species – ring cleavage reactions. J Gen Microbiol 135:1957–1967Google Scholar
  60. 60.
    Bell SG, Harford-Cross CF, Wong LL (2001) Engineering the CYP101 system for in vivo oxidation of unnatural substrates. Protein Eng 14:797–802CrossRefPubMedGoogle Scholar
  61. 61.
    Blake JAR, Pritchard M, Ding SH, Smith GCM, Burchell B, Wolf CR, Friedberg T (1996) Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli. FEBS Lett 397:210–214CrossRefPubMedGoogle Scholar
  62. 62.
    Gillam EMJ, Wunsch RM, Ueng YF, Shimada T, Reilly PEB, Kamataki T, Guengerich FP (1997) Expression of cytochrome P450 3A7 in Escherichia coli: effects of 5′ modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase. Arch Biochem Biophys 346:81–90CrossRefPubMedGoogle Scholar
  63. 63.
    Kim D, Ortiz de Montellano PR (2009) Tricistronic overexpression of cytochrome P450cam, putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system. Biotechnol Lett 31:1427–1431CrossRefPubMedCentralPubMedGoogle Scholar
  64. 64.
    Parikh A, Gillam EMJ, Guengerich FP (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat Biotechnol 15:784–788CrossRefPubMedGoogle Scholar
  65. 65.
    Schneider S, Wubbolts MG, Sanglard D, Witholt B (1998) Biocatalyst engineering by assembly of fatty acid transport and oxidation activities for in vivo application of cytochrome P-450BM-3 monooxygenase. Appl Environ Microbiol 64:3784–3790PubMedCentralPubMedGoogle Scholar
  66. 66.
    Schneider S, Wubbolts MG, Sanglard D, Witholt B (1998) Production of chiral hydroxy long chain fatty acids by whole cell biocatalysis of pentadecanoic acid with an E. coli recombinant containing cytochrome P450BM-3 monooxygenase. Tetrahedron Asymmetry 9:2833–2844CrossRefGoogle Scholar
  67. 67.
    Peterson JA (1971) Camphor binding by Pseudomonas putida cytochrome P450. Arch Biochem Biophys 144:678–693CrossRefGoogle Scholar
  68. 68.
    Atkins W, Sligar S (1989) Molecular recognition in cytochrome P450 − Alteration of regioselective alkane hydroxylation via protein engineering. J Am Chem Soc 111:2715–2717CrossRefGoogle Scholar
  69. 69.
    Trudgill PW (1990) Microbial metabolism of monoterpenes – recent developments. Biodegradation 1:93–105CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Jeanette E. Stok
    • 1
  • Kate E. Slessor
    • 1
  • Anthony J. Farlow
    • 1
  • David B. Hawkes
    • 1
  • James J. De Voss
    • 1
    Email author
  1. 1.School of Chemistry and Molecular BiosciencesUniversity of QueenslandBrisbaneAustralia

Personalised recommendations