Substrate Recognition and Dynamic Transport in MHETase Reveals Regulation Mechanism of Gatekeeper Residues: a Theoretical Investigation

Abstract

Polyethylene terephthalate (PET) is a widely used plastic, but its waste pollution has become a serious global environmental concern. The discovery of PETase offers a green and sustainable approach for PET degradation. However, the accumulation of intermediate products generated by PETase can inhibit enzyme activity. MHETase exhibits extremely high specificity for the intermediate product MHET, which is further hydrolyzed into ethylene glycol (EG) and terephthalic acid (TPA). In this study, we employed a combination of theoretical simulation techniques to elucidate the substrate binding mode and transport mechanism of MHETase at the atomic level. Our results reveal that substrate recognition by MHETase depends primarily on interactions with the carboxylic acid group and aromatic ring of MHET, and destroying these interactions significantly weakens the substrate specificity of MHETase. Residues Phe415, Gln410, and Trp397 act as gatekeepers, facilitating substrate binding while alleviating steric hindrance from the side chain of Phe415. This work provides a comprehensive analysis of the structural and functional features of MHETase, identifying critical residues and binding modes. These findings offer valuable insights for enzyme engineering aimed at improving the performance of PET hydrolysis enzymes.