Synthesis of 19‐Hydroxyarachidonic Acid by Fungal Peroxygenases: An Experimental and Computational Study

Hydroxylated derivatives of arachidonic acid play crucial roles in physiology and inflammation response, with distinct biological effects for (R)- and (S)-enantiomers, highlighting their pharmaceutical relevance. Here, recombinant unspecific peroxygenases (UPOs) are harnessed-from the fungi Coprinopsis cinerea (rCciUPO) and Agrocybe aegerita (rAaeUPO), and the rAaeUPO engineered variants A77L, A77T, and A77N-to regio- and enantio-selectively hydroxylate arachidonic acid. rCciUPO and rAaeUPO primarily produced ω-1 and ω-2 hydroxy derivatives, with certain overoxidation to keto forms, while the rAaeUPO variants show enhanced regioselectivity to ω-1 hydroxylation and minor overoxidation. Remarkably, A77L exhibited very good enantioselectivity, yielding 92% of the (S)-enantiomer of 19-hydroxyarachidonic acid. Molecular dynamics simulations revealed that the narrowing of the active-site channel by the A77L mutation imposes critical torsional constraints on the substrate, favoring selective hydroxylation and preventing overoxidation of hydroxylated products. In this way, key interactions involving residues T242, E245, and D70 modulate enantioselectivity by interacting with the substrate's carboxylate moiety. Additional experimental assays show that these UPOs efficiently hydroxylate other bioactive C18-C22 fatty acids. The experimental and computational data integration findings provide a rational basis for engineering UPO selectivity, presenting the enzyme variant A77L as a promising biocatalyst for the selective synthesis of pharmacologically relevant hydroxy-fatty acids.