Chen_2026_J.Hazard.Mater_517_143488

Phthalic acid esters (PAEs) are persistent pollutants with environmental and health concerns. Dialkyl/monoalkyl PAEs hydrolases can fully degrade PAEs, offering sustainable bioremediation potential, yet their bifunctional mechanism remains unclear. This study resolved co-crystal structures of EstJ6 bound to catalytic intermediate, monobutyl phthalate (MBP) and its hydrolytic end product, phthalic acid (PA), revealing a conserved alpha/beta hydrolase fold. Molecular dynamics simulations and crystallographic B-factor comparisons indicate that the catalytic core is largely pre-stabilized into a productive conformation prior to substrate binding, while the lid domain retains dynamic plasticity. Although the catalytic triad (composed of Ser146, His270, and Glu240) mediates hydrolysis of dibutyl phthalate (DBP) to MBP and then PA, the second step is inefficient. Structural analysis revealed an excessive nucleophilic attack distance (6.1-6.4 A) between Ser146 and the second ester bond, beyond the optimal 3-4 A range. Moreover, inadequate electrostatic shielding within EstJ6's negatively charged tunnel further compromises MBP binding stability. Mutagenesis identified Trp176 as crucial for Pi-Pi stacking and Leu274 as a steric hindrance. Notably, the L274A mutant showed 1.5-fold higher activity, suggesting tunnel engineering as a viable optimization strategy. These findings advance the mechanistic understanding of PAEs hydrolases for bioremediation applications.