Novel enzymes involved in the biotransformation of oxytetracycline by <i>Arthrobacter nicotianae</i> OTC-16

Current research on the microbial degradation of antibiotic residues primarily focuses on isolating degrading bacteria and characterizing their metabolites. The enzymatic mechanisms underlying these biotransformation processes remain poorly understood. Here, we investigated the molecular mechanisms involved in the biodegradation of oxytetracycline (OTC) by <i>Arthrobacter nicotianae</i> OTC-16 using integrated transcriptomic and proteomic sequencing analyses. OTC exposure significantly altered gene expression, identifying 1,158 and 863 differentially expressed genes on days 2 and 4, respectively. Proteomics revealed 352 and 818 differentially expressed proteins, predominantly involved in compound binding and redox processes. Integrated analysis identified <i>AlkB</i> (DNA oxidative demethylase), <i>uaZ</i> (urate oxidase), and <i>prpD</i> (2-methylcitrate dehydratase) as key enzymes mediating OTC biodegradation through decarboxylation, carbon-carbon bond reduction, and dehydration reactions, confirmed by RT-qPCR. Protein-protein interactions subsequently highlighted supportive proteins, including glutathione S-transferase, ABC transporter ATP-binding protein, prpB, prpE, MarR regulators, and glyoxalase. Notably, <i>prpD</i> and <i>uaZ</i> were successfully expressed in <i>Escherichia coli</i>, and their OTC degradation-promoting activities were validated through <i>in vitro</i> assays. This study is the first to report the roles of <i>prpD</i> and <i>uaZ</i> in antibiotic residue degradation, providing novel insights into the enzymatic mechanisms and laying a foundation for future genetic engineering and practical applications.IMPORTANCEOxytetracycline (OTC), a widely used antibiotic in agriculture and animal husbandry, frequently accumulates in the environment, posing significant ecological and human health risks by promoting antibiotic resistance. Understanding the microbial enzymatic pathways for OTC biodegradation is crucial for developing effective bioremediation strategies. This study provides a comprehensive molecular analysis of the OTC degradation process by <i>Arthrobacter nicotianae</i> OTC-16, identifying three key enzymes (<i>AlkB</i>, <i>uaZ</i>, and <i>prpD</i>) involved in OTC removal. Successfully expressing <i>prpD</i> and <i>uaZ</i> in <i>Escherichia coli</i> and validating their biodegradation activity <i>in vivo</i> further underscores their potential for biotechnological applications. These findings significantly enhance the knowledge of microbial antibiotic degradation mechanisms, offering practical molecular targets for environmental detoxification strategies.