Skip to main content
SpringerLink
Log in

Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Solomon EI, Sundaram UM, Machonkin TE (1996) Chem Rev 96:2563–2605

    Article  PubMed  CAS  Google Scholar 

  2. Stoj CS, Kosman DJ (2005) In: King RB (ed) Encyclopedia of inorganic chemistry, vol II, 2nd edn. Wiley, New York, pp 1134–1159

  3. Lindley PF (2001) In: Bertini I, Sigel A, Sigel H (eds) Handbook on metalloproteins. Dekker, New York, pp 763–811

    Google Scholar 

  4. Blair DF, Campbell GW, Schoonover JR, Chan SI, Gray HB, Malmstrom BG, Pecht I, Swanson BI, Wooddruff WH, Cho WK, English AM, Fry HA, Lum V, Norton KA (1985) J Am Chem Soc 107:5755–5766

    Article  CAS  Google Scholar 

  5. Martins LO, Soares CM, Pereira MM, Teixeira M, Jones GH, Henriques AO (2002) J Biol Chem 277:18849–18859

    Article  PubMed  CAS  Google Scholar 

  6. Hullo M-F, Moszer I, Danchin A, Martin-Verstraete I (2001) J Bacteriol 183:5426–5430

    Article  PubMed  CAS  Google Scholar 

  7. Donovan W, Zheng L, Sandman K, Losick R (1987) J Mol Biol 196:1–10

    Article  PubMed  CAS  Google Scholar 

  8. Enguita FJ, Martins LO, Henriques AO, Carrondo MA (2003) J Biol Chem 278:19416–25

    Article  PubMed  CAS  Google Scholar 

  9. Bento I, Martins LO, Gato GL, Carrondo MA, Lindley PF (2005) Dalton Trans 21:3507– 3513

    Article  PubMed  Google Scholar 

  10. Durão P, Bento I, Fernandes AT, Melo EP, Lindley PF, Martins LO (2006) J Biol Inorg Chem 11:514–526

    Article  PubMed  Google Scholar 

  11. Davis-Kaplan SR, Askwith CC, Bengtzen AC, Radisky D, Kaplan J (1998) Proc Natl Acad Sci USA 95:13641–13645

    Article  PubMed  CAS  Google Scholar 

  12. Blackburn NJ, Ralle M, Hassett R, Kosman DJ (2000) Biochemistry 39:2316–2324

    Article  PubMed  CAS  Google Scholar 

  13. Palmer AE, Szilagyi RK, Cherry JR, Jones A, Xu F, Solomon EI (2003) Inorg Chem 42:4006–4017

    Article  PubMed  CAS  Google Scholar 

  14. Hellman NE, Kono S, Mancini GM, Hoogeboom AJ, de Jong GJ, Gitlin JD (2002) J Biol Chem 277:46632–46638

    Article  PubMed  CAS  Google Scholar 

  15. Galli I, Musci G, di Patti MCB (2004) J Biol Inorg Chem 9:90–95

    Article  PubMed  CAS  Google Scholar 

  16. Xu F (1999) In: Flickinger MC, Drewn SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis and bioseparation. Wiley, New York, pp 1545–1554

  17. Brenner AJ, Harris ED (1995) Anal Biochem 226:80–84

    Article  PubMed  CAS  Google Scholar 

  18. Bradford MM (1976) Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  19. Aasa R, Väangard VT (1975) J Magnet Reson 19:308–315

    CAS  Google Scholar 

  20. Sanchez-Amat A, Lucas-Elio P, Fernández E, Garcia-Borrón JC, Solano F (2001) Biochim Biophys Acta 1547:104–116

    PubMed  CAS  Google Scholar 

  21. Grass G, Rensing C (2003) FEMS Microbiol Rev 27:197–213

    Article  PubMed  Google Scholar 

  22. Changela A, Chen K, Xue Y, Holschen J, Outten CE, O’Halloran TV, Mondragón A (2003) Science 301:1383–1387

    Article  PubMed  CAS  Google Scholar 

  23. Finney LA, O’Halloran TV (2003) Science 300:931–936

    Article  PubMed  CAS  Google Scholar 

  24. Outten FW, Huffman DL, Hale JA, O’Halloran TV (2001) J Biol Chem 276:30670–30677

    Article  PubMed  CAS  Google Scholar 

  25. Macomber L, Rensing C, Imlay JA (2007) J Bacterial 189:1616–1626

    Article  CAS  Google Scholar 

  26. Beswick PH, Hall GH, Hook AJ, Little K, McBride DCH, Lott KAK (1976) Chem Biol Interact 14:347–356

    Article  PubMed  CAS  Google Scholar 

  27. Partdridge JD, Sanguinetti G, Dibden D, Roberts RE, Poole RK, Green J (2007) J Biol Chem 282:11230–11237

    Article  Google Scholar 

  28. Green MT (2006) J Am Chem Soc 128:1902–1906

    Article  PubMed  CAS  Google Scholar 

  29. Palmer AE, Randall DW, Xu F, Solomon EI (1999) J Am Chem Soc 121:7138–7149

    Article  CAS  Google Scholar 

  30. Machokin TE, Quintanar L, Palmer AE, Hassett R, Severance S, Kosman DJ, Solomon EI (2001) J Am Chem Soc 123:5507–5517

    Article  Google Scholar 

  31. Kataoka K, Kitagawa R, Inoue M, Naruse D, Sakurai T, Huang H-W (2005) Biochemistry 44:7004–7012

    Article  PubMed  CAS  Google Scholar 

  32. Volkin DB, Klibanov AM (1989) In: Creighton TE (ed) Minimizing protein inactivation protein function. A practical approach. IRL, Oxford, pp 1–24

    Google Scholar 

  33. Savini I, D’Alessio S, Giartosio A, Morpurgo L, Avigliano L (1990) Eur J Biochem 190:491–495

    Article  PubMed  CAS  Google Scholar 

  34. di Patti MCB, Musci G, Giartosio A, D’Alessio S, Calabrese L (1990) J Biol Chem 265:21016–21022

    Google Scholar 

  35. Vassall KA, Stathopulos PB, Rumfeldt JAO, Lepock JR, Meiering EM (2006) Biochemistry 45:7366–7379

    Article  PubMed  CAS  Google Scholar 

  36. Agostinelli E, Cervoni L, Giartosio A, Morpurgo L (1995) Biochem J 306:697–702

    PubMed  CAS  Google Scholar 

  37. Ragusa S, Cambria MT, Pierfederici F, Scirè A, Bertoli E, Tanfani F, Cambria A (2002) Biochim Biophys Acta 1601:155–162

    PubMed  CAS  Google Scholar 

  38. Koroleva OV, Stepanova EV, Binukov VI, Timofeev VP, Pfeil W (2001) Biochimic Biophys Acta 1547:397–407

    CAS  Google Scholar 

  39. Milardi D, Grasso DM, Verbeet MP, Canters GW, La Rosa C (2003) Arch Biochem Biophys 414:121–127

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by POCI/BIO/57083/2004 and FP6-2004-NMP-NI-4/026456 project grants. P.F. Lindley and A. Sanchez Amat are acknowledged for their useful suggestions. We thank P. Jackson for correcting the English. Z. Chen holds a Post-doc fellowship (SFRH/BPD/27104/2006) and A.T. Fernandes a PhD fellowship (SFRH/BPD/31444/2006).

Author information

Authors and Affiliations

  1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. Da República, 2781-901, Oeiras, Portugal

    Paulo Durão, Zhenjia Chen, André T. Fernandes, Smilja Todorovic, Manuela M. Pereira & Lígia O. Martins

  2. Sekr. PC14, Max-Volmer-Laboratorium für Biophysikalische Chemie, Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 10623, Berlin, Germany

    Peter Hildebrandt & Daniel H. Murgida

  3. Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

    Eduardo P. Melo

  4. Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Lisbon, Portugal

    Eduardo P. Melo

Authors
  1. Paulo Durão
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhenjia Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. André T. Fernandes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter Hildebrandt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniel H. Murgida
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Smilja Todorovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Manuela M. Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Eduardo P. Melo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lígia O. Martins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lígia O. Martins.

Additional information

Paulo Durão and Zhenjia Chen contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Durão, P., Chen, Z., Fernandes, A.T. et al. Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes. J Biol Inorg Chem 13, 183–193 (2008). https://doi.org/10.1007/s00775-007-0312-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-007-0312-0

Keywords

Search

Navigation