title: Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.
abstract: The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å 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, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °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 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °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.
authors.affiliation: Department of Material and Life Science, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Sintawee
authors.initials: S
authors.lastname: Sulaiman
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: You
authors.forename: Eiko
authors.initials: E
authors.lastname: Kanaya
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Koga
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007700:Kinetics; D054892:Metagenome / Q000235:genetics*; D011093:Polyethylene Terephthalates / Q000737:chemistry; D011489:Protein Denaturation; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D011093:Polyethylene Terephthalates; D002265:Carboxylic Ester Hydrolases; C019952:cutinase