Exploring substrate promiscuity of unspecific peroxygenase from Agrocybe aegerita by neutral genetic drift

Abstract

Unspecific peroxygenase (UPO, EC.1.11.2.1) is a heme-thiolate peroxidase with exclusive mono(per)oxygenase activity and plenty of potential applications in organic synthesis. Fuelled by catalytic amounts of H2O2, UPO acts as a self-sufficient mono-oxygenase with a complex catalytic mechanism that joins the reactive intermediates of heme-peroxidases and P450s (“peroxide shunt” pathway). Thus, the versatile peroxide-dependent monooxygenase activity of UPO based on a two-electron oxygenation mechanism, permits an array of reactions to occur, among them: bromide oxidation, sulfoxidation, N-oxidation, aromatic peroxygenation, double bond epoxidation, hydroxylation of aliphatic compounds and ether cleavages [1, 2]. In a recent work, our laboratory performed the first protein engineering study carried out on UPO. After 5 generations of molecular evolution in Saccharomyces cerevisiae, we were able to produce a highly active, soluble and stable enzyme that is readily secreted in yeast [3]. With the view of taking advantage of this evolutionary platform, in this communication, the evolved UPO variant has been subjected to several rounds of neutral genetic drift to explore the substrate promiscuity and the evolvability of this versatile enzyme.