No abstract

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

Ethers can be found in the environment as structural, active or even pollutant molecules, although their degradation is not efficient under environmental conditions. Fungal unspecific heme-peroxygenases (UPO were reported to degrade low-molecular-weight ethers through an H2O2-dependent oxidative cleavage mechanism. Here, we report the oxidation of a series of structurally related aromatic ethers, catalyzed by a laboratory-evolved UPO (PaDa-I) aimed at elucidating the factors influencing this unusual biochemical reaction. Although some of the studied ethers were substrates of the enzyme, they were not efficiently transformed and, as a consequence, secondary reactions (such as the dismutation of H2O2 through catalase-like activity and suicide enzyme inactivation) became significant, affecting the oxidation efficiency. The set of reactions that compete during UPO-catalyzed ether oxidation were identified and quantified, in order to find favorable conditions that promote ether oxidation over the secondary reactions.