The role of phenylalanines in the access channel of surface displayed unspecific peroxygenase from Agrocybe aegerita

• 15 variants of Aae UPO PaDa-I were successful produced and characterised. • Established yeast surface display as screening system for unspecific peroxygenases. • Mutations resulted in increased NBD activity and altered ABTS/NBD activity ratios. • Falsified the causation of substrate preference of unspecific peroxygenases being caused by nature of amino acids in access channel. • Identified the effect of phenylalanine substitutions in the access channel of Aae UPO PaDa-I and the importance of every phenylalanine for the catalysis of cyclohexane hydroxylation. Unspecific peroxygenases are an emerging class of oxyfunctionalization enzymes with a broad substrate spectrum. The structure of their access channel, that allows substrates to enter the catalytic heme center, depends on the enzyme family. Short UPOs access channel is typically lined with aliphatic amino acids like leucine and isoleucine while long UPOs access channel is mostly lined with aromatic phenylalanines. The type of amino acids in the access channel aligns with the preferred substrate class for each enzyme family. In this study we exchanged each of the seven phenylalanines in the access channel of the best researched enzyme in this class, the unspecific peroxygenase from Agrocybe aegerita , with aliphatic amino acids and investigated how the substrate preference of the enzyme variants compares to the wildtype enzyme. For 15 enzyme variants resulting from substitutions of three phenylalanines in close proximity to the heme center, we conducted docking studies. The results supported our hypothesis, that the substrate preference would shift towards aliphatic substrates when phenylalanines are exchanged with aliphatic amino acids. Additionally, we constructed 35 mutant strains of the production host Komagataella phaffii for the expression and surface display of enzyme variants for all phenylalanines in the enzyme´s access channel. Characterization of these enzyme variants revealed that substrate preference changed opposite to the hypothesis and docking results, decreasing activity for aliphatic substrates and increasing activity for aromatic ones. We concluded that steric conditions in the active site are more important to the enzyme´s substrate preference then the nature of the amino acids and identified the most important residues for different substrates.