A novel insight of enhancing the hydrogen peroxide tolerance of unspecific peroxygenase from Daldinia caldariorum based on structure

Abstract

Unspecific peroxygenases (UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert CH bonds using H2O2 as an oxygen donor without cofactors. However, the enhancement of the H2O2 tolerance of UPOs is always challenging. In this study, the A161C mutant of rDcaUPO, which originates from Daldinia caldariorum, was found to be highly H2O2-resistant. Compared with the wild type, the mutant rDcaUPO-A161C showed a 10-h prolonged half-life and a 64% improved enzyme activity when incubated in 10 mmol/L H2O2. The crystal structure analysis at 1.47 Å showed that rDcaUPO-A161C exhibited 10 α-helixes (cyan) and a series of ordered rings, forming a single asymmetric spherical structure. The two conserved domains near heme formed an active site with the catalytic PCP and EHD regions (Glu86, His87, Asp88 residues). The H2O2 tolerance of rDcaUPO-A161C was preliminarily explored by comparing its structure with the wild type. Notably, rDcaUPO-A161C showed significantly higher catalytic efficiency than the wild type for the production of hydroxyl fatty acids. This study is anticipated to provide an insight into the structure-function relationship and expand potential applications of UPOs.