The Molecular Mechanism of Chloramphenicol and Thiamphenicol Resistance Mediated by a Novel Oxidase CmO in Sphingomonadaceae

Antibiotic resistance mediated by bacterial enzyme inactivation plays a mysterious and crucial role for antibiotic degradation and selection pressure reduction in the environment. The enzymatic inactivation of the antibiotic chloramphenicol (CAP) involves nitro reduction, amide bond hydrolysis and acetylation modification. However, the molecular mechanism of enzymatic oxidation of CAP remains unknown. Here, a novel oxidase gene cmO was identified and confirmed biochemically to catalyze the resistance process through the oxidative inactivation at the side chain C-3’ position of CAP and thiamphenicol (TAP) in Sphingomonadaceae. The oxidase CmO is highly conservative in Sphingomonadaceae and shares the highest amino acid homology of 41.05% with the biochemically identified glucose methanol choline (GMC) oxidoreductases. Molecular docking and site-directed mutagenesis analyses demonstrated that CAP was anchored inside the protein pocket of CmO with the hydrogen bonding of key residues glycine (G)99, asparagine (N)518, methionine (M)474 and tyrosine (Y)380. CAP sensitivity test demonstrated that the acetyltransferase and CmO showed higher resistance to CAP as compared with the amide bond-hydrolyzing esterase and nitroreductase. This study provides a better theoretical basis and a novel diagnostic gene for understanding and assessing the fate and resistance risk of CAP and TAP in the environment.ImportanceRising levels of antibiotic resistance undermines ecological and human health as a result of indiscriminate usage of antibiotics. Various resistance mechanisms have been revealed, for instance genes encoding proteins that degrade antibiotics, yet requiring further exploration. In this study, we reported a novel gene encoding an oxidase involved in the inactivation of typical amphenicol antibiotics (chloramphenicol and thiamphenicol), and the molecular mechanism was elucidated. The observation provides novel data to understand capabilities of bacteria to tackle antibiotic stress and suggests complex function of enzymes in the context of antibiotic resistance development and antibiotics removal. The reported gene can be further employed as an indicator to monitor amphenicols fate in the environment, benefiting the risk assessment in this era of antibiotic resistance.