Abstract: Phenolic acid decarboxylase (PAD, EC 4.1.1.102) specifically catalyzes irreversible decarboxylation of phenolic acids, producing 4-vinyl derivatives, which have great application value in the fields of food flavor enhancement and biocatalysis. The substantial variation in enzymatic properties of PADs produced by different strains restricts their precise application. Accordingly, exploration and systematic enzymatic characterization of novel PADs from specific strains are of great significance for expanding the PAD resource library and advancing their practical application. In this study, a PAD gene from Lactiplantibacillus plantarum RS20D was heterologously expressed, yielding the recombinant enzyme LpPAD. LpPAD exhibited the highest specific activity and catalytic efficiency toward caffeic acid. Molecular dynamics simulations revealed that the binding of LpPAD to caffeic acid led to a more stable conformation of the enzyme as well as optimized active-site flexibility and structural complementarity. Isothermal titration calorimetry (ITC) further demonstrated that there was a strong synergistic effect in the binding process of caffeic acid, with the lowest dissociation constant and highest entropic contribution, indicating superior binding affinity and reaction driving force. These findings provide structural insights into the substrate specificity of LpPAD toward caffeic acid, establishing a theoretical framework for the directed modification of PAD and the industrial-scale bioproduction of 4-vinylcatechol.

Key words: phenolic acid decarboxylase; caffeic acid; enzymatic properties; molecular dynamics simulation; isothermal titration calorimetry