An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca²⁺ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca²⁺ and Zn²⁺, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open, and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca²⁺ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.

中文翻译：

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Ca2 +调节的PET水解酶Cut190的多个结构状态及其与活性和稳定性的关系。

来自嗜热分离株Saccharomonospora viridis AHK190的酶Cut190可解聚聚对苯二甲酸乙二醇酯（PET）。Cut190及其S226P / R228S突变体Cut190 *的催化活性和稳定性受Ca ²⁺结合的调节。我们先前确定了Cut190 *，Cut190 * S176A非活性突变体与金属离子，Ca ²⁺和Zn ²⁺形成复合物的晶体结构。，以及底物，琥珀酸单乙酯和己二酸单乙酯。在这项研究中，我们确定了另一个Cut190 *突变体Cut190 **的晶体结构，其中Cut190 *的三个C末端残基被删除，并且非活性突变体Cut190 ** S176A与金属离子形成了络合物。除了先前观察到的闭合，打开和接合形式外，我们还确定了喷射形式，这将使产品不可逆地解离，然后进行下一个反应周期。这些多种形式可能是受Ca ²⁺调节的Cut190的稳定或亚稳态。结合，并与酶的功能密切相关。在删除C-末端残基后，我们发现在保持活性的同时，热稳定性提高了。增加的稳定性可用于Cut190的蛋白质工程中以进行PET解聚，因为它需要在PET的玻璃化转变温度以上进行反应。