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Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes
Zitatschlüssel ISI:000252673400003
Autor Durao, Paulo and Chen, Zhenjia and Fernandes, Andre T. and Hildebrandt, Peter and Murgida, Daniel H. and Todorovic, Smilja and Pereira, Manuela M. and Melo, Eduardo P. and Martins, Ligia O.
Seiten 183-193
Jahr 2008
ISSN 0949-8257
DOI 10.1007/s00775-007-0312-0
Adresse 233 SPRING STREET, NEW YORK, NY 10013 USA
Journal J. Biol. Inorg. Chem.
Jahrgang 13
Nummer 2
Monat FEB
Verlag SPRINGER
Zusammenfassung 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.
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