Xing_2026_Chem.Eng.J__173543

Acetylcholinesterase (AChE) is a key enzyme in neural signaling, exhibiting exceptional specificity for the hydrolysis of acetylcholine. Microbial acetylesterases with AChE-like activity hold considerable potential for biotechnological applications. Here, we report a novel GDSL-type acetylesterase from Aspergillus niger GZUF36 (AnAE) that displays confirmed AChE-like activity. Structural analyses of wild-type AnAE and its N267D and S34A mutants, complexed with substrate analogs (acetate, propionate) or pesticides, reveal striking differences from animal AChEs in overall fold, active-site architecture, catalytic triad configuration, and oxyanion-hole geometry. The enzyme's negatively charged, bowl-shaped pocket facilitates the recognition of choline esters. Combined crystallographic, docking, and mutagenesis studies identify the acyl pocketparticularly the gating residue Tyr269as critical for acetylcholine specificity. These findings provide (i) a structural framework for designing minimalist acetylesterases with AChE-like activity and (ii) a microbial model for high-throughput pesticide screening.