Intermolecular interactions mediate the stimulatory and inhibitory effects of typical veterinary antibiotics on soil xylanase activity

Veterinary antibiotic residues are increasingly detected in the soil environment, therefore raising concerns about their effects on soil health. Xylanases are key soil extracellular enzymes responsible for hemicellulose degradation and organic carbon turnover; however, their responses to antibiotic exposure, particularly at the level of direct intermolecular interactions, remain poorly understood. Considering their extensive use and frequent detection in soil media, sulfadiazine (SD) and tetracycline hydrochloride (TC) were chosen as representative veterinary antibiotics for this study. The molecular mechanisms underlying the effects of the SD, TC, and SD + TC mixtures on the enzymatic activity of endo -β-1,4-xylanase (XYL) were investigated using various spectroscopy techniques, interactive effect assessments, and molecular docking studies. The results revealed that SD inhibited XYL activity in a concentration-dependent manner, whereas TC increased XYL activity in a dose-dependent manner. These alterations in XYL activity were mediated by spontaneous antibiotic–enzyme interactions at a 1:1 stoichiometric ratio, rather than by the induction of significant structural perturbations in the enzyme. Furthermore, SD and TC were found to target distinct regulatory sites in XYL, which may explain their opposite effects: SD interacted with the catalytic center, while TC bound to the dimer interface. Molecular modeling further visualized and corroborated the interaction behaviors of the tested antibiotics at their respective binding sites. Moreover, the interactive effect of the SD + TC mixture on the XYL activity shifted from synergistic to antagonistic when the SD-to-TC ratio decreased, indicating that TC binding at the inter-subunit interface might directly modulate the binding behavior of SD. This study establishes a methodological framework for exploring the intermolecular interactions between typical veterinary antibiotics and soil biomacromolecules, thereby providing critical evidence for determining the molecular-scale effects of antibiotic contaminants and facilitating precision risk assessment in the soil environment.