Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.
Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
Proteins. 2021 May;89(5):502-511. doi: 10.1002/prot.26034. Epub 2020 Dec 24.
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 -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 Cut190SS, and its inactive S176A mutant, Cut190SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190S176A 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 Cut190SS series were different from those of the Cut190* series. However, our results show that binding of Ca to the weak binding site, site 1, would be retained, enabling Cut190SS to keep its ability to use Ca to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190SS 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.
来自嗜热菌 Saccharomonospora viridis AHK190 的角质酶样酶 Cut190 是一种有效降解聚对苯二甲酸乙二醇酯 (PET) 的候选酶。我们之前开发了 Cut190 的突变体 (S226P/R228S)，我们将其命名为 Cut190*，该突变体具有更高的活性和稳定性，并解决了其晶体结构。最近，我们发现一个 Ca 结合位点上的 D250C/E296C 突变导致热稳定性提高，同时保留其聚酯酶活性。在这项研究中，我们解析了 Cut190突变体 Q138A/D250C-E296C/Q123H/N202H 的晶体结构，命名为 Cut190SS，及其无活性的 S176A 突变体 Cut190SS_S176A，分辨率很高。整体结构与之前报道的 Cut190和 Cut190S176A 相似。正如预期的那样，Cys250 和 Cys296 紧密相邻形成二硫键，这肯定有助于提高稳定性。等温滴定量热实验和 3D 参考相互作用位点模型计算表明，Cut190SS 系列的金属结合特性与 Cut190系列不同。然而，我们的结果表明，结合到弱结合位点 1 的 Ca 仍将被保留，使 Cut190SS 能够保持利用 Ca 加速构象从封闭（无活性）到开放（活性）形式的变化的能力。在提高热稳定性的同时，Cut190SS 仍能表达其酶功能。即使在 70°C 孵育，相当于 PET 的玻璃化转变温度，酶仍保持良好的活性，这意味着 Cut190SS 具有很高的用于工业 PET 解聚的适用性。
