Functional Characterization and Catalytic Mechanism of an Amidase Involved in Acetaminophen Degradation from Acinetobacter sp.

The accumulation of acetaminophen (APAP) in agricultural soils and water systems poses risks to ecosystems and public health. This study characterizes Acinetobacter sp. DL27, an APAP-degrading strain with broad temperature and pH adaptability, demonstrating its bioremediation potential in soil and wastewater. Through high-resolution mass spectrometry, we identified three novel metabolic intermediates, thereby refining the bacterial APAP degradation pathway. Multiomics analysis elucidated metabolic coordination and stress-tolerance mechanisms, leading to the identification of a novel amidase (AdA). Recombinant AdA exhibited activity over 10-60 °C and pH 4.0-10.0, with a Km of 8.96 ± 1.03 μM and a kcat/Km of 9.04 μM-1s-1, indicating higher catalytic efficiency than previously reported APAP amidases. Molecular dynamics simulations and site-directed mutagenesis confirmed that a Ser161-Ser185-Lys82 triad constitutes the catalytic center driving amide cleavage. These findings provide mechanistic insights into bacterial APAP biodegradation and highlight the practical application potential of strain DL27 and AdA.