Insights into the Interaction Mechanism of Protocatechuate 3,4-Dioxygenase with Substrates via Multispectroscopy and Molecular Docking

Protocatechuic acid (PCA) is a key intermediate in aromatic pollutant biodegradation, and protocatechuate 3,4-dioxygenase (3,4-PCD) can efficiently cleave its aromatic ring. The interaction mechanism remains incompletely understood. This study investigated the interaction mechanism between PCA and a 3,4-PCD from <i>Rhodococcus</i> sp. 2G. The recombinant enzyme was heterologously expressed in <i>Escherichia coli</i>, yielding 2.7 mg/L with high catalytic efficiency. Fluorescence quenching confirmed static quenching and efficient complex formation. Multispectral analysis revealed conformational and microenvironmental changes upon PCA binding. Integrated molecular dynamics simulations and site-directed mutagenesis identified Tyr253, Tyr337, His389, His391, and Arg386 as synergistic residues for PCA ring cleavage, with hydrogen bonds and electrostatic interactions being crucial. A refined catalytic mechanism was proposed. These findings provide deep biophysical insights into 3,4-PCD/PCA interactions, advance mechanistic knowledge of dioxygenases in aromatic catabolism, and offer a basis for enzyme engineering to reduce pollutants in agricultural and food systems.