Mechanistic insight into esterase-catalyzed hydrolysis of phthalate esters (PAEs) based on integrated multi-spectroscopic analyses and docking simulation

Abstract

A novel PAE-hydrolyzing esterase (named Hyd) gene was screened from the genomic library of Rhodococcus sp. 2G and was successfully expressed in heterologous E. coli, which was defined as a new family of esterolytic enzymes. The purified Hyd could efficiently degrade various PAEs, displaying high activity and stability with a broad range of pH (4-10) and temperature (20-60 °C). Interaction mechanism of Hyd with dibutyl phthalate (DBP) was investigated by integrated multi-spectroscopic and docking simulation methods. Fluorescence and UV-vis spectra revealed that DBP could quench the fluorescence of Hyd through a static quenching mechanism. The results from synchronous fluorescence and CD spectra confirmed that the DBP binding to Hyd triggered conformational and micro-environmental changes of Hyd, which were characterized by increased stretching extent and random coil, and decreased α-helix and β-sheet. Molecular docking study showed that DBP could be bound to the cavity of Hyd with hydrogen bonding and hydrophobic interaction. A novel and distinctive catalytic mechanism was proposed: two key residues Thr¹⁹⁰ and Ser¹⁹¹ might catalyze the hydrolysis of DBP, instead of the conserved catalytic triad (Ser-His-Asp) reported elsewhere, which was confirmed by site-directed mutagenesis.

Keywords: Enzyme catalysis; Molecular docking; Multi-spectroscopic analyses; Phthalate hydrolase; Site-directed mutagenesis.