The effect of double (S238F/W159H) mutations on the structure and dynamics of PET degrading enzyme

Polyethylene terephthalate (PET) is one of the highly produced synthetic polymers worldwide and had acquired attention due to its impact resistance, high clarity, and light weight. PET has become the first choice in making disposable bottles, leading to massive scales of production resulting in very high utilization across various facets of our daily life. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation, thus presenting a serious threat to the ecosystem. Degradation of PET by enzymatic hydrolysis is a promising strategy to depolymerize the PET into its monomers. In recent studies, a plastic-degrading enzyme known as PETase (IsPETase) from the Ideonella sakaiensis has been identified to hydrolyze PET. The wild-type enzyme from Ideonella sp., has been engineered to improve the catalytic activity. While the IsPETase and its variants have been the subject of extensive structural and biochemical studies, the corresponding computational studies to support the improved activity of the mutant enzyme is not fully understood. In this work, we employed all-atom classical molecular dynamics simulations of the wild-type and double mutant IsPETase enzymes to investigate the underlying reason for the improved catalytic activity in the double mutant by means of structure-dynamics-function relationship. Our results show that the engineered mutations reshape the active site structure, volume, and dynamics of the protein loops which is crucial for substrate binding. We also demonstrate that addition of aromatic and hydrogen bond-forming residues near catalytic site improves binding affinity. This work will enable the rational design of mutants for enhanced PET degrading activity.

聚对苯二甲酸乙二酯（Polyethylene terephthalate, PET）是全球范围内产量极高的合成聚合物之一，凭借出色的抗冲击性、高透明度与轻质特性受到广泛关注。PET已成为一次性水瓶的首选材料，大规模生产使其在日常生活诸多领域得到极高程度的应用。然而，PET以废弃物形式大量积累，且极难生物降解，对生态系统构成严重威胁。采用酶促水解法降解PET，是将其解聚为单体的极具前景的策略。近年来的研究发现，从坂崎伊德菌（Ideonella sakaiensis）中分离得到的PET降解酶PETase（IsPETase）可水解PET。研究人员已对伊德菌属野生型该酶进行工程化改造以提升催化活性。尽管IsPETASE及其变体已被广泛开展结构与生化特性研究，但阐明突变酶活性提升机制的相关计算研究仍相对不足。本研究针对野生型与双突变型IsPETase酶开展全原子经典分子动力学模拟，通过结构-动态-功能关联关系探究双突变体催化活性提升的内在机制。研究结果表明，工程化突变重塑了对底物结合至关重要的蛋白质环区的活性位点结构、体积与动态特性。本研究还证实，在催化位点附近引入芳香族残基与可形成氢键的残基，可提升酶与底物的结合亲和力。本研究将为理性设计具备更强PET降解活性的突变体提供理论指导。