Structure-guided engineering of a cold-adapted 2,4-dichlorophenol hydroxylase for enhanced low-temperature...

Chlorophenolic compounds are persistent hazardous pollutants whose removal is particularly challenging in cold environments due to reduced microbial and enzymatic activity. In this study, a cold-adapted 2,4-dichlorophenol hydroxylase (TfdB-JLU) was systematically investigated and engineered to enhance pollutant degradation efficiency at low temperatures. Comparative structural analysis with homologous enzymes revealed key features associated with cold adaptation, including reduced stabilizing interactions, increased internal cavities, and enhanced flexibility in loop regions proximal to the catalytic pocket. Molecular dynamics simulations further demonstrated that localized flexibility facilitates substrate accommodation and catalytic turnover under low-temperature conditions. Guided by these insights, a structure-based engineering strategy was implemented through targeted glycine substitutions to increase conformational adaptability. The engineered variants exhibited significantly improved catalytic performance at 4°C, with specific activities increased by up to 1.79-fold (0.82 U/mg) compared with the wild-type enzyme. Kinetic analysis revealed reduced K_m and enhanced k_cat, while thermodynamic evaluation indicated decreased activation free energy barriers, supporting improved catalytic efficiency at 4°C. Importantly, the engineered enzymes achieved markedly enhanced degradation of 2,4-dichlorophenol and related chlorophenolic compounds under low-temperature conditions. This work elucidates the structural determinants underlying cold adaptation of aromatic monooxygenases and demonstrates an effective strategy for improving enzymatic pollutant removal in cold environments. The engineered TfdB variants provide promising biocatalysts for low-temperature bioremediation of chlorophenolic contaminants.