Temperature-Dependent Active-Site Rearrangements of PETaseSM14: Insights from Molecular Dynamics Simulations

Polyethylene terephthalate (PET) is a synthetic polymer that is widely used in the production of textiles, packaging materials, and beverage bottles. However, its high durability and resistance to abiotic degradation result in serious environmental and health problems. PETase is an enzyme that can depolymerize PET into value-added products, thereby providing an environmentally friendly strategy for PET recycling. PETaseSM14 from a marine sponge, Streptomyces sp. SM14, has a high salt tolerance and thermal stability, thus suggesting its potential for PET degradation applications. However, the substrate recognition mechanism of PETase remains unclear because the catalytic residue is buried within residues that form the substrate-binding cleft. To elucidate the molecular mechanism of PETaseSM14, all-atom molecular dynamics simulations were performed at 300, 320, and 340 K. The results revealed that the overall α/β fold remained stable at all temperatures, whereas temperature-dependent local fluctuations and conformational changes were observed in the substrate-binding cleft and N-terminal region. At 300 and 320 K, positional shifts and conformational changes in Tyr88 exposed the catalytic Ser156 to the solvent, thereby forming a potential substrate-binding cleft. In contrast, at 340 K, which is higher than the melting temperature of PETaseSM14, disruption of the charge-relay system of the catalytic triad occurs through conformational changes in His234. Substantial temperature-dependent conformational and positional changes in the N-terminal region of PETaseSM14 were observed at 320 and 340 K. These results provide mechanistic insight into the temperature-dependent active-site rearrangements and offer rational engineering strategies to enhance the efficiency of PETase for PET biodegradation.