Abstract: As a biocatalyst, phenolic acid decarboxylase catalyzes the decarboxylation of its substates into 4-vinyl derivatives, which exhibit numerous advantages and holds promising prospects. In this study, phenolic acid decarboxylase from Bacillus subtilis J6 (BJ6PAD) was engineered by C-terminal substitution to obtain mutants with enhanced catalytic capacity. Homology modeling, molecular docking, and molecular dynamics simulation were employed to analyze the interactions between the enzyme and its substrates. The results indicated that C-terminal substitution affected the enzyme’s substrate affinity and catalytic efficiency. The constructed mutants, BJ6PAD-C1 and BJ6PAD-C2, exhibited significantly improved catalytic activity towards most phenolic acid substrates, particularly caffeic acid, towards which their activities increased by 20.34% and 29.97% when compared with that of the wild-type enzyme. Moreover, both mutants showed increased affinity for ferulic acid, caffeic acid, and sinapic acid. Specifically, the catalytic efficiency of BJ6PAD-C1 increased by 4%, 24%, and 145% for these three phenolic acids, respectively, and that of BJ6PAD-C2 by 7%, 37%, and 114%, respectively. Finally, molecular docking analysis predicted the key binding sites of the substrates, revealing that C-terminal substitution modulated the interactions between the key amino acid residues in the substrate-binding cavity. Molecular dynamics simulation was performed to elucidate the reasons for the altered catalytic activity from two perspectives: protein structure and energy differences, suggesting that the mutations might widen the protein import channel, facilitating easier ligand access to the substrate-binding pocket. Meanwhile, structural changes in the enzyme could lead to off-target effects of substrates, thereby reducing its catalytic activity. This study not only enriches PAD resources but also offers new insights into the relationship between the structure and function of PAD.

Key words: phenolic acid decarboxylase; C-terminal substitution; enzymatic properties; molecular docking; molecular dynamics simulation