Structural Rigidity and Nucleophilic Proximity Synergistically Enable Efficient High-Temperature Hydrolysis of Phthalate Esters by PestE

Abstract Phthalic acid esters (PAEs) are persistent environmental pollutants. Their industrial biodegradation demands thermostable, efficient hydrolases. We characterized PestE, a thermostable esterase hydrolyzing both dialkyl and monoalkyl PAEs. PestE retained >98% of its maximal activity in short-term assays at 80–100 °C, with minimal loss after 12 h, and retained ∼50% activity after 48 h at 80 °C, with 99.4% dibutyl phthalate degradation in 2 min. Molecular dynamics simulations indicated that hydrophobic core reorganization and increased surface hydrophilicity synergistically enhance thermostability by balancing global rigidity and local flexibility. Co-crystallization with monobutyl phthalate revealed a substrate-guiding tunnel and catalytic machinery (Ser157-His284-Asp254 triad and oxyanion hole Gly85/Gly86/Ala158) for two-step dealkylation. A key feature is the close proximity of the nucleophilic Ser157 to the substrate carbonyl carbon (2.8–3.1 Å), enabling efficient catalysis. Mutagenesis confirmed key hydrophobic residues for substrate binding and active-site stability. These findings establish PestE as a promising biocatalyst for industrial PAE degradation.