Structure-guided directed evolution of deacetylase leads to increased specificity of chloroacetamides and effective production of chloroamines

O-3-chloro-2-propenyl hydroxylamine (chloroamine) is the primary material for the production of cyclohexenone herbicides, such as clethodim, which are widely used in agricultural production due to their high efficiency, low toxicity, and good environmental safety. In this study, a bioenzymatic strategy is proposed for the synthesis of chloroamines in order to address the polluting nature of traditional chemical synthesis. Using easily synthesized chloroacetamides as the model substrate, histone deacetylase 10 (zHDAC10) from zebrafish was used as the biocatalyst for the deacetylation process, resulting in the production of chloroamides. Through a structure-directed mutagenesis and site saturation mutagenesis strategy, a zHDAC10-M2 (A24RE274K) was created, leading to approximately an 8-fold enhancement of activity in comparison to the zHDAC10-WT, with a high catalytic efficiency of 40 mM and a 24-hour conversion of over 94% on a 100-mL preparative scale. Structural analysis and molecular dynamics showed that the mutation of 24 and 274 sites to positively charged amino acids enabled them to form hydrogen bonds with the terminal Cl of chloroamines, thereby stabilizing the state of the substrate in the active pocket and thus improving the catalytic efficiency. The biosynthetic route established in this study provides a new promising way for the production of chloroamines.