Structural Elucidation and Mechanisms-Guided Engineering of a Promiscuous Esterase for Enhanced Polyurethane Depolymerization
Jiawei Liu, Mingna Zheng, Yuan Wen, Wei Xia, Xu Han, Jie ZHOU, Weidong Liu, Ren Wei, Yanwei Li, Weiliang Dong, Min Jiang
Abstract
Polyurethane (PU) is highly resistant to biodegradation, primarily due to the intrinsic stability of its urethane bond. Aside from a small number of amidases reported to hydrolyze (poly)urethane bonds, several promiscuous esterases have also been found to catalyze PU degradation. In this study, we clarified the ligand-free crystal structure of Aes72, an esterase enzyme that exhibits promiscuous hydrolytic activity toward carbamate and amide bonds, at a high resolution of 1.80 Å. We investigated the catalytic mechanism underlying urethane bond cleavage by Aes72 using multiscale quantum mechanics/molecular mechanics (QM/MM) simulations. Our findings indicate that the reaction mechanism consists of four concerted elementary steps, with the nucleophilic attack (step i) identified as the rate-determining step. The subsequent structure-guided engineering of Aes72 yielded several enhanced single mutants, ultimately resulting in a superior double mutant, F276A/L141I. This variant exhibited approximately a two-fold increase in catalytic efficacy toward bis(4-hydroxybutyl) (methylenebis(4,1-phenylene)) dicarbamate (BMC) hydrolysis and significantly enhanced degradation performance on two distinct polyether-based PU materials compared to the wild-type enzyme. Our findings provide essential mechanistic insights into the structure–function relationship of the promiscuous esterase Aes72 in PU degradation and demonstrate its potential applicability in bio-based plastic recycling.