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.

中文翻译：

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一种新型氧化酶CmO介导的鞘氨醇对氯霉素和甲砜霉素耐药的分子机制

由细菌酶失活介导的抗生素耐药性在环境中抗生素降解和选择压力降低方面起着神秘而关键的作用。抗生素氯霉素 (CAP) 的酶失活涉及硝基还原、酰胺键水解和乙酰化修饰。然而，CAP的酶促氧化的分子机制仍然未知。在这里，一种新的氧化酶基因 cmO 被鉴定和生化证实，通过在鞘氨醇科中 CAP 和甲砜霉素 (TAP) 的侧链 C-3' 位置的氧化失活来催化抗性过程。氧化酶 CmO 在鞘氨醇单胞菌科中高度保守，与生化鉴定的葡萄糖甲醇胆碱 (GMC) 氧化还原酶具有 41.05% 的最高氨基酸同源性。分子对接和定点诱变分析表明，CAP 通过关键残基甘氨酸 (G)99、天冬酰胺 (N)518、蛋氨酸 (M)474 和酪氨酸 (Y)380 的氢键锚定在 CmO 的蛋白质袋内。CAP敏感性试验表明，与酰胺键水解酯酶和硝基还原酶相比，乙酰转移酶和CmO对CAP表现出更高的抗性。该研究为了解和评估CAP和TAP在环境中的命运和耐药风险提供了更好的理论基础和新的诊断基因。CAP敏感性试验表明，与酰胺键水解酯酶和硝基还原酶相比，乙酰转移酶和CmO对CAP表现出更高的抗性。该研究为了解和评估CAP和TAP在环境中的命运和耐药风险提供了更好的理论基础和新的诊断基因。CAP敏感性试验表明，与酰胺键水解酯酶和硝基还原酶相比，乙酰转移酶和CmO对CAP表现出更高的抗性。该研究为了解和评估CAP和TAP在环境中的命运和耐药风险提供了更好的理论基础和新的诊断基因。