Structure-guided evolution of Marinobacter subterrani deacetylase for enhanced herbicide precursor synthesis by substrate-binding pocket and channels engineering.

Deacetylases can directly hydrolyze amide bonds for the environmentally friendly synthesis of the herbicide intermediate O-(3-chloro-2-propenyl)hydroxylamine. However, the rigid halogenated chain of N-[(E)-3-chloroprop-2-enoxy]acetamide (NECA) may hinder productive substrate positioning and limit catalytic efficiency. Accordingly, 20 deacetylases were initially screened, and a deacetylase from Marinobacter subterrani (MsDeac) was identified as the optimal starting scaffold. To improve its catalytic performance toward NECA, a synergistic pocket and tunnel reshaping strategy was subsequently implemented to modify the substrate-binding environment and predicted access pathway. Molecular docking suggested a putative halogen-bonding interaction in M3 that may contribute to substrate positioning. M3 also exhibited an expanded predicted pocket volume (from 777 to 1055 Å3) and an increased number of predicted access channels. After stepwise mutagenesis, a triple mutant (Y325H-M321H-Y170H, M3) was successfully obtained, demonstrating a 68-fold increase in catalytic efficiency (kcat/Km), with an increased conversion of 80 mM NECA from approximately 10% to > 99.5% within 12 h. The results suggest that improved spatial complementarity and a potential halogen-bonding interaction may contribute to enhanced catalytic efficiency, providing structural hypotheses for the engineering of deacetylases toward sterically challenging substrates.