Characterization of Two 4,5-PCD Isoenzymes Identified from Curvibacter sp. PAE-UM

Phthalates (PAEs), such as dibutyl phthalate (DBP), are emerging contaminants in agriculture that are commonly used in plastic films, pesticides, and soil amendments. Their accumulation in water threatens ecosystems and human health. Biodegradation offers a promising solution, but the DBP degradation mechanisms remain unclear. This study identified two 4,5-PCD isoenzymes (PCD34 and PCD67) in Curvibacter sp. PAE-UM. PCD34 showed higher affinity and broader substrate specificity, while PCD67 had better thermal stability and metal ion sensitivity. Both enzymes converted protocatechuate (PCA) to 4CHMS at 45% efficiency in 5 min. Molecular dynamics revealed the stable binding of PCD67, while mutagenesis enhanced PCD34 activity (124.4%) by reducing steric hindrance. These findings may aid in the engineering of strains for PAE bioremediation in agricultural wastewater.