Abstract
The accumulation of polyethylene terephthalate (PET), a widely used polyester plastic in packaging and textiles, has led to a global environmental crisis. Biodegradation presents a promising strategy for PET recycling, with PET hydrolases (PETase) undertaking the task at the molecular level. Unfortunately, PETase operates only at ambient temperatures with low efficiency, limiting its industrial application. Current engineering efforts focus on enhancing the thermostability of PETase, but increased stability can reduce the structural dynamics needed for substrate binding, potentially slowing enzymatic activity. To elucidate the balance between stability and flexibility in optimizing PETase catalytic activity, we performed theoretical investigations on both wild-type PETase (WT-PETase) and a thermophilic variant (Thermo-PETase) using molecular dynamics simulations and frustration analysis. Despite being initially designed to stabilize the native structure of the enzyme, our findings reveal that Thermo-PETase exhibits an unprecedented increase in structural flexibility at the PET-binding and catalytic sites, beneficial for substrate recruitment and product release, compared to WT-PETase. Upon PET binding, we observed that the structural dynamics of Thermo-PETase is largely quenched, favoring the proximity between the catalytic residues and the carbonyl of the PET substrate. This may potentially contribute to a higher probability of a catalytic reaction occurring in Thermo-PETase compared to WT-PETase. We suggest that Thermo-PETase can exhibit higher PET-degradation performance than WT-PETase across a broad temperature range by leveraging stability and flexibility at high and low temperatures, respectively. Our findings provide valuable insights into how PETase optimizes its enzymatic performance by balancing stability and flexibility, which may contribute to future PETase design strategies.