Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes.
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.
Durão
Paulo
P
Universidade Nova de Lisboa, Av. Da República, 2781-901, Oeiras, Portugal.
Chen
Zhenjia
Z
Fernandes
André T
AT
Hildebrandt
Peter
P
Murgida
Daniel H
DH
Todorovic
Smilja
S
Pereira
Manuela M
MM
Melo
Eduardo P
EP
Martins
Lígia O
LO
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D003300:Copper; D042845:Laccase; D010100:Oxygen