In contrast to the well-studied mammalian acetylcholinesterases (AChE), the structures and molecular mechanism of prokaryotic acetylcholinesterases have just started to be uncovered. We have recently reported the first structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa (Pham VD et al Shi R, JBC 2020). Drastically different from the mammalian enzymes, ChoE adopts a typical fold of the SGNH-hydrolase family constituting an interesting example of convergent evolution. Combined with kinetic analyses of WT and mutant enzymes, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. These results indicate that substrate inhibition in ChoE is due to acetate product release being blocked by the binding of a substrate molecule in a nonproductive mode (i.e., formation of an E-P-S complex). In addition, our most recent (unpublished) results reveal three distinct conformations of Ser38 highlighting an unprecedented dynamics of the Ser residue in the catalytic triad. The observed flexibility of Ser38 is well correlated with the functional requirement of various catalytic steps and may indicate a general feature for the hydrolases containing the Asp-His-Ser catalytic triad. Furthermore, using a mutant enzyme, we have captured an enzyme-intermediate-substrate (E-I-S) ternary complex (unpublished) in which a substrate molecule is bound on top of an acyl intermediate covalently linked to Ser38. This molecular snapshot of a rarely experimentally observed phenomenon significantly advances our overall understanding of enzyme catalysis and inhibition. Taken together, our work has provided novel insights into the bacterial acetylcholinesterases, active site dynamics in the SGNH-family hydrolases and general enzyme catalysis and inhibition.