Elucidation of the crystal structure of Coriolopsis caperata laccase: restoration of the structure and activity of the native enzyme from the T2-depleted form by copper ions

Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. A new laccase was isolated from the basidiomycete Coriolopsis caperata strain 0677 and its amino-acid sequence was determined. According to its physicochemical properties and spectroscopic features, the laccase from C. caperata is a high redox-potential blue laccase. Attempts to crystallize the native enzyme were unsuccessful. The copper type 2-depleted (T2D) laccase was prepared and crystallized. The structure of T2D laccase from C. caperata was solved at 1.6 angstrom resolution, and attempts to reconstruct the T2 copper centre were performed using Cu+ and Cu2+ ions. The structure of T2D+Cu+ laccase was solved at 1.89 angstrom resolution. It was shown thatthe T2D+Cu+ laccase structure contained four copper ions in the active site. Reconstruction could not be achieved when the T2D laccase crystals were treated with CuSO4.