LeSaint_2026_Biosci.Rep__

Aldicarb is a carbamate pesticide used for pest control in agriculture. As a fast-acting acetylcholinesterase inhibitor, aldicarb interferes with the nervous system by preventing the breakdown of acetylcholine. Aldicarb could be used as a chemical-warfare agent to cause mass casualty incidents. There is no specific FDA approved medication for aldicarb detoxification.Our previous study revealed that a Fc-fused butyrylcholinesterase (BChE) mutant, known as CocH3-Fc(M3), can be inhibited rapidly by aldicarb and that the aldicarb-inhibited enzyme CocH3-Fc(M3) can be reactivated spontaneously, suggesting that CocH3-Fc(M3) may hydrolyze aldicarb. However, the suggested CocH3-Fc(M3)-catalyzed hydrolysis of aldicarb was not confirmed experimentally in the previous study. In the present study, by developing a LC-MS/MS method to detect and quantify aldicarb and aldicarb oxime concentrations, we were able to directly observe the CocH3-Fc(M3)-catalyzed aldicarb hydrolysis for the first time, confirming that CocH3-Fc(M3) indeed has the desirable catalytic activity for aldicarb hydrolysis and may be considered as the first aldicarb hydrolase identified so far. Further, we carried out Michaelis-Menten kinetic analysis on the CocH3-Fc(M3)-catalyzed aldicarb hydrolysis and determined the catalytic parameters (kcat=0.060 min-1, KM=2.5microM, and kcat/KM=2.4x10min-1 M-1) at 37oC. The obtained kinetic parameters at 37oC will be valuable for further in vivo studies and translational research using CocH3-Fc(M3) and for designing more potent enzymes to hydrolyze aldicarb in the future. Additionally, the LC-MS/MS method developed in this study may serve as a valuable tool to accurately detect aldicarb and its reaction products in future food and environmental safety control efforts and aldicarb-related toxicology studies.