4EB0

Crystal structure of Leaf-branch compost bacterial cutinase homolog

4EB0 の概要

• エントリーDOI: 10.2210/pdb4eb0/pdb
• 分子名称: LCC, THIOCYANATE ION (3 entities in total)
• 機能のキーワード: hydrolase, serine esterase, cutinase homolog, pet degradation, metagenome
• 由来する生物種: uncultured bacterium
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 28032.32

構造登録者

Sulaiman, S.,You, D.J.,Eiko, K.,Koga, Y.,Kanaya, S. (登録日: 2012-03-23, 公開日: 2013-03-27, 最終更新日: 2026-03-18)

主引用文献

Sulaiman, S.,You, D.J.,Kanaya, E.,Koga, Y.,Kanaya, S.
Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.
Biochemistry, 53:1858-1869, 2014

PubMed 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.

PubMed: 24593046
DOI: 10.1021/bi401561p

実験手法

X-RAY DIFFRACTION (1.5 Å)