Unveiling the analgesic and antipyretic drug acetaminophen catabolic mechanism in Pseudomonas taiwanensis AP-1

Acetaminophen (APAP), an analgesic and antipyretic drug, is commonly detected in wastewater treatment plant (WWTP) effluents, surface water, and soil, indicating its status as an emerging environmental contaminant. In this study, we isolated a bacterium, Pseudomonas taiwanensis AP-1, capable of completely mineralizing APAP and utilizing it as the sole carbon source for growth. A newly identified metabolite, γ-glutamyl-4-aminophenol (γ-G4AP), was reported for the first time in the degradation of APAP by strain AP-1. Two amidases (ApaH1 and ApaH2), responsible for the conversion of APAP to 4-aminophenol (4-AP), were identified through a combination of genomic comparison, heterologous expression, and gene knockout. Notably, ApaH1 played a pivotal role in the degradation of APAP by strain AP-1. The catalytic triad of ApaH1 (K82-S161-S185) and ApaH2 (K85-S160-S184) were identified as by molecular docking and site-directed mutagenesis. Additionally, a gene cluster apd for the metabolism of 4-AP was also successfully identified in strain AP-1, consisting of the aniline dioxygenase gene cluster apdBCD1D2EF and the BT catabolic gene apdGH . Interestingly, the 4-AP metabolic gene cluster apd was highly conserved among other Pseudomonas strains capable of APAP degradation. Our results provide new insights into the mechanism of APAP biodegradation and strain AP-1 may be a promising bacterium for the bioremediation of APAP pollutions.