Structural basis of mutants of PET‐degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2021-05, Vol.89 (5), p.502-511
• Main Authors: Emori, Miho, Numoto, Nobutaka, Senga, Akane, Bekker, Gert‐Jan, Kamiya, Narutoshi, Kobayashi, Yuma, Ito, Nobutoshi, Kawai, Fusako, Oda, Masayuki
• Format: Article
• Language: English
• Subjects: Binding sites; Calcium ions; Calcium-binding protein; Calorimetry; Crystal structure; Cutinase; Depolymerization; disulfide bond; enzymatic activity; Enzymes; Glass transition temperature; metal binding; Mutants; Mutation; Polyethylene; Polyethylene terephthalate; Thermal stability; Titration; Titration calorimetry; Transition temperatures
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.26034

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 Ca2+‐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 Ca2+ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca2+ 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.