1. Structural basis of mutants of <scp>PET</scp> ‐degrading enzyme from Saccharomonospora viridis <scp>AHK190</scp> with high activity and thermal stability
- Author
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Narutoshi Kamiya, Nobutaka Numoto, Masayuki Oda, Miho Emori, Akane Senga, Nobutoshi Ito, Fusako Kawai, Yuma Kobayashi, and Gert-Jan Bekker
- Subjects
Models, Molecular ,Conformational change ,Hot Temperature ,Protein Conformation ,Stereochemistry ,Genetic Vectors ,Mutant ,Gene Expression ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Bacterial Proteins ,Structural Biology ,Enzyme Stability ,Escherichia coli ,Protein Interaction Domains and Motifs ,Thermal stability ,Cysteine ,Disulfides ,Cloning, Molecular ,Binding site ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,Polyethylene Terephthalates ,Depolymerization ,Hydrolysis ,Thermophile ,030302 biochemistry & molecular biology ,Isothermal titration calorimetry ,Recombinant Proteins ,Actinobacteria ,Enzyme ,chemistry ,Mutation ,Calcium ,Environmental Pollutants ,Carboxylic Ester Hydrolases ,Protein Binding - 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 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.
- Published
- 2020