Structural basis of mutants of PET‐degrading enzyme from Saccharomonospora viridisAHK190 with high activity and thermal stability

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.

Structural basis for Ca²⁺-dependent catalysis of a cutinase-like enzyme and its engineering: application to enzymatic PET depolymerization

“…However, increasing stability by mutations could result in significantly decreased activity in many cases, mainly because the naïve conformational change, including fluctuation, would be critical for activity [11]. In Cut190, Asp250 and Glu296 are located close to each other, and the Cys substitutions form a disulfide bond, which was confirmed by crystal structure analysis [12]. Upon mutation, T m increased by 23°C [7].…”

“…The activities of Cut190* toward PBSA in the presence of 0.25 mM Mn 2+ and 2.5 mM Mg 2+ were approximately 90% and 40% of that in the presence of 2.5 mM Ca 2+ , respectively, whereas that in the presence of Zn 2+ was markedly lower[16]. The results indicated that the order of binding affinity at Site 1 was Ca 2+ < Mg 2+ < Mn 2+ < Zn2+ .ITC experiments revealed no binding heats of metal ions, even Zn 2+ , for Cut190*SS and Cut190*SS_S176A, in which disulfide bonds were introduced at residues 250 and 296, close to Site 2[12]. Upon mutation, not only Site 2, but also other metal-binding sites would be perturbed for metal binding.…”

“…Challenging the goal of PET recycling using enzymes is important and contributes to basic science in a wide range of fields, such as protein chemistry and polymer chemistry. and other data are taken from previous papers [12,16,20].…”

“…The catalytic activity of S226P/R228S mutant was 1.5-fold higher than that of S226P mutant. Therefore, the mutant S226P/R228S designated as Cut190* was used as a template to introduce further mutations to increase its activity and thermal stability ( Table 1 ) [ 6 – 8 ]. Upon the S176A mutation on Cut190*, no activity was observed even at a concentration 20-fold higher than that of Cut190*.…”

“…Although the activity of Cut190*SS toward PBSA at 37°C was similar to that of Cut190*, the residual activity after incubating the enzyme at 70°C was significantly enhanced [ 6 , 10 ]. The residual activities of Cut190* and Cut190*SS after incubation at 70°C for 2 h were approximately 10% and 95% relative to the activity without incubation, respectively [ 6 ]. These results strongly indicate that Cut190*SS is a promising candidate for PET degradation.…”