Computational Redesign of a Urethanase for Efficient Polyurethane Depolymerization

Polyurethane
(PU) is one of the most widely used petroleum-based
plastics, significantly contributing to the global plastic waste crisis.
In recent years, enzyme-based recycling technology has emerged as
a promising eco-friendly solution to plastic pollution. Several urethanases
capable of hydrolyzing PU have recently been identified. However,
their low activity limits their utility for efficient PU degradation.
Herein, we investigated the mechanism of PU hydrolysis catalyzed by
the urethanase UMG-SP1 through multiscale quantum mechanics/molecular
mechanics calculations using a substrate mimic containing two carbamate
bonds. The deacylation stage was identified as the rate-determining
step with an estimated Gibbs free energy barrier of 19.0 kcal·mol–1, consistent with experimentally determined range
of 16.8–16.9 kcal·mol–1. The effect
of active-site structure and the enzymatic electric field on catalytic
activity was analyzed, and their relationships with catalytic efficiency
were established. Based on these relationships, we proposed two semirational
enzyme-engineering strategies that successfully identified beneficial
mutations. Ultimately, we obtained several UMG-SP1 mutants with improved
hydrolytic activities, including the most active variant, L126M, which
exhibits a close to 6-fold improvement in depolymerizing a self-synthesized
thermoplastic polyether-PU compared to the wild-type enzyme. These
findings offer a semirational approach for urethanase engineering,
which has substantial potential for extension to the development of
other plastic hydrolases.