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.

中文翻译：

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具有高活性和热稳定性的绿色糖单胞菌 AHK190 PET 降解酶突变体的结构基础

来自嗜热菌Saccharomonospora viridis AHK190的类角质酶Cut190 是有效解聚聚对苯二甲酸乙二醇酯 (PET) 的良好候选者。我们之前开发了一个 Cut190 (S226P/R228S) 的突变体，我们将其命名为 Cut190*，它既提高了活性又提高了稳定性，并解决了其晶体结构。最近，我们发现 D250C/E296C 在 Ca ²⁺之一上的突变结合位点导致更高的热稳定性，同时保持其聚酯酶活性。在本研究中，我们以高分辨率解析了 Cut190* 突变体 Q138A/D250C-E296C/Q123H/N202H（命名为 Cut190*SS）及其非活性 S176A 突变体 Cut190*SS_S176A 的晶体结构。整体结构与之前报道的 Cut190* 和 Cut190*S176A 的结构相似。正如预期的那样，Cys250 和 Cys296 位置紧密以形成二硫键，这肯定有助于增加稳定性。等温滴定量热实验和 3D 参考相互作用位点模型计算表明，Cut190*SS 系列的金属结合特性与 Cut190* 系列的不同。然而，我们的结果表明 Ca ^{2+ 的}结合到弱结合位点，位点 1，将被保留，使 Cut190*SS 能够保持其使用 Ca ²⁺加速从封闭（非活性）到开放（活性）形式的构象变化的能力。在提高热稳定性的同时，Cut190*SS 仍能发挥其酶促作用。即使在 70°C（对应于 PET 的玻璃化转变温度）下孵育后，该酶仍能很好地保持其活性，这意味着使用 Cut190*SS 进行工业 PET 解聚具有很高的适用性。