Structure–function relationships in unspecific peroxygenases revealed by a comparative study of their action on the phenolic lignin monomer 4-propylguaiacol

Unspecific peroxygenases (UPOs) are versatile enzymes capable of oxidizing a broad range of substrates, using hydrogen peroxide as the sole co-substrate. In this study, UPOs were evaluated for their potential in the selective oxyfunctionalization of the phenolic lignin monomer 4-propylguaiacol (4-PG) to generate versatile scaffolds for the synthesis of high-value compounds. In addition to the desired peroxygenase reaction, the phenolic group of 4-PG is susceptible to undesirable one-electron oxidation (peroxidase activity). Assessment of the activity of 19 UPOs from phylogenetically diverse clades toward 4-PG revealed that several UPOs could serve as potential biocatalysts for the functionalization of 4-PG, with some enzymes showing both promising conversion yields (>50%) and regioselectivity for the peroxygenase reaction. Pronounced differences in peroxygenase:peroxidase activity ratios and regioselectivity were observed. Comparative analysis—supported by experimental activity profiles and structural data—suggest that a more constrained active-site topology contributes to the peroxygenase activity. UPOs from a clade within the Ascomycota phylum with high peroxygenase activity possess a unique aliphatic pocket in their catalytic centers. Our study provides valuable insights into the structure–function relationships underpinning enhanced peroxygenase activity of UPOs and provides a functional mapping of a broad UPO-sequence space for 4-PG, highlighting these enzymes as promising catalysts for the selective oxyfunctionalization of a phenolic lignin monomer.