Molecular Origin of the Enhanced PET Degradation Activity of LCC-ICCG Revealed by Computational Modeling

Poly(ethylene terephthalate) (PET) hydrolases have emerged as promising biocatalysts for closed-loop plastic recycling. Among the most efficient enzymes reported to date, LCC-ICCG exhibits exceptional PET-depolymerization performance under industrially relevant conditions. However, the molecular basis for its superior activity relative to engineered PETases such as FAST-PETase and HotPETase remains incompletely understood. Here, we combine microsecond-scale molecular dynamics simulations, quantum mechanical cluster calculations, pre-reaction-state analysis, noncovalent-interaction mapping, and distortion/interaction activation strain analysis to compare LCC-ICCG with FAST-PETase and HotPETase. The simulations show that LCC-ICCG samples catalytically competent pre-reaction-state geometries more frequently, mainly because V212 reshapes the local environment around the scissile ester. This residue relieves steric congestion, supports weak - C–H···O guided substrate preorganization, and reinforces both the Asp-His catalytic dyad and the W190-associated pocket architecture. Density functional theory calculations further indicate that this preorganized active site lowers the acylation barrier to 15.5 kcal/mol by reducing substrate distortion and strengthening transition-state interactions. High-temperature simulations show that LCC-ICCG better preserves near-attack geometries at 350 K, linking thermal robustness to sustained catalytic preorganization. Moreover, reciprocal I208V mutations in IsPETase-derived enzymes enrich pre-reaction-state populations, supporting the transferability of the V212-centered design principle. Overall, these results establish pre-reaction-state stabilization as a key determinant of PET-hydrolase efficiency and provide mechanistic design rules for engineering next-generation PET depolymerases.