7CTR

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

(2021) Proteins 89: 502-511

• PubMed: 33340163 Search on PubMed
• DOI: https://doi.org/10.1002/prot.26034
• Primary Citation of Related Structures:  
  7CTR, 7CTS


• 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 ²⁺ -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 ²⁺ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca ²⁺ 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.

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Organizational Affiliation: 

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.