Abstract
Unspecific peroxygenases(UPOs,EC 1.11.2.1)is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C-H bonds using H2O2 as an oxygen donor without cofactors.However,the enhance-ment 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 ac-tivity when incubated in 10mmol/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 cat-alytic 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.
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