Unraveling the Interplay between Stability and Flexibility\nin the Design of Polyethylene Terephthalate (PET) Hydrolases

The accumulation\nof polyethylene terephthalate (PET), a widely\nused polyester plastic in packaging and textiles, has led to a global\nenvironmental crisis. Biodegradation presents a promising strategy\nfor PET recycling, with PET hydrolases (PETase) undertaking the task\nat the molecular level. Unfortunately, PETase operates only at ambient\ntemperatures with low efficiency, limiting its industrial application.\nCurrent engineering efforts focus on enhancing the thermostability\nof PETase, but increased stability can reduce the structural dynamics\nneeded for substrate binding, potentially slowing enzymatic activity.\nTo elucidate the balance between stability and flexibility in optimizing\nPETase catalytic activity, we performed theoretical investigations\non both wild-type PETase (WT-PETase) and a thermophilic variant (Thermo-PETase)\nusing molecular dynamics simulations and frustration analysis. Despite\nbeing initially designed to stabilize the native structure of the\nenzyme, our findings reveal that Thermo-PETase exhibits an unprecedented\nincrease in structural flexibility at the PET-binding and catalytic\nsites, beneficial for substrate recruitment and product release, compared\nto WT-PETase. Upon PET binding, we observed that the structural dynamics\nof Thermo-PETase is largely quenched, favoring the proximity between\nthe catalytic residues and the carbonyl of the PET substrate. This\nmay potentially contribute to a higher probability of a catalytic\nreaction occurring in Thermo-PETase compared to WT-PETase. We suggest\nthat Thermo-PETase can exhibit higher PET-degradation performance\nthan WT-PETase across a broad temperature range by leveraging stability\nand flexibility at high and low temperatures, respectively. Our findings\nprovide valuable insights into how PETase optimizes its enzymatic\nperformance by balancing stability and flexibility, which may contribute\nto future PETase design strategies.