Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase

The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 angstrom resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T-1/2, and that of the GdnHCl-induced unfolding curve, C-m, at 30 degrees C were 86.2 degrees C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, Delta G(H2O), was 41.8 kJ mol(-1) at 30 degrees C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, k(u)(H2O), of 3.28 x 10(-6) s(-1) at 50 degrees C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 degrees C) was considerably lower than the T-1/2 value. It increased by 10 degrees C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 degrees C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.