7CTS

Open form of PET-degrading cutinase Cut190 with thermostability-improving mutations of S226P/R228S/Q138A/D250C-E296C/Q123H/N202H and S176A inactivation

7CTS の概要

• エントリーDOI: 10.2210/pdb7cts/pdb
• 分子名称: Alpha/beta hydrolase family protein, 1,4-DIETHYLENE DIOXIDE, BICINE, ... (5 entities in total)
• 機能のキーワード: protein engineering, polyesterase, disulfide bond, metal binding, hydrolase
• 由来する生物種: Saccharomonospora viridis
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 29180.89

構造登録者

Emori, M.,Numoto, N.,Senga, A.,Bekker, G.J.,Kamiya, N.,Ito, N.,Kawai, F.,Oda, M. (登録日: 2020-08-20, 公開日: 2021-02-03, 最終更新日: 2024-11-06)

主引用文献

Emori, M.,Numoto, N.,Senga, A.,Bekker, G.J.,Kamiya, N.,Kobayashi, Y.,Ito, N.,Kawai, F.,Oda, M.
Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.
Proteins, 89:502-511, 2021

PubMed Abstract: The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS.

PubMed: 33340163
DOI: 10.1002/prot.26034

実験手法

X-RAY DIFFRACTION (1.1 Å)