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JBIC Journal of Biological Inorganic Chemistry

, Volume 13, Issue 2, pp 183–193 | Cite as

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

  • Paulo Durão
  • Zhenjia Chen
  • André T. Fernandes
  • Peter Hildebrandt
  • Daniel H. Murgida
  • Smilja Todorovic
  • Manuela M. Pereira
  • Eduardo P. Melo
  • Lígia O. MartinsEmail author
Original Paper

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.

Keywords

CotA laccase Multicopper oxidases Copper incorporation Copper homeostasis 

Notes

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).

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Copyright information

© SBIC 2007

Authors and Affiliations

  • Paulo Durão
    • 1
  • Zhenjia Chen
    • 1
  • André T. Fernandes
    • 1
  • Peter Hildebrandt
    • 2
  • Daniel H. Murgida
    • 2
  • Smilja Todorovic
    • 1
  • Manuela M. Pereira
    • 1
  • Eduardo P. Melo
    • 3
    • 4
  • Lígia O. Martins
    • 1
    Email author
  1. 1.Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal
  2. 2.Sekr. PC14, Max-Volmer-Laboratorium für Biophysikalische Chemie, Institut für ChemieTechnische Universität BerlinBerlinGermany
  3. 3.Centro de Biomedicina Molecular e EstruturalUniversidade do AlgarveFaroPortugal
  4. 4.Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica e QuímicaInstituto Superior TécnicoLisbonPortugal

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