Characterization of Recombinant Unspecific Peroxygenase from Candolleomyces aberdarensis Through Crystallographic and Substrate Selectivity Studies

Fungal unspecific peroxygenases (UPOs) are remarkable biocatalysts for the selective oxygenation of non-activated C─H bonds. Here, we describe a crystallographic and substrate selectivity study of an UPO ortholog from Candolleomyces (Psathyrella) aberdarensis (PabUPO-II). The recombinant enzyme produced in yeast was crystallized and complexed with a representative panel of substrates, including alkanes, fatty acids, and norisoprenoids; the crystals diffracted at a resolution up to 2 Å. PabUPO-II combines structural features of canonical long and short UPOs, presenting a hybrid heme channel and a flexible catalytic Glu212 that adopts two alternate conformations, proximal and distal to the substrates. The positioning of substrates at the heme channel in soaking experiments was complemented with a characterization of the enzymatic reactions. With alkanes and fatty acids, PabUPO-II carried out oxygenations at ω-2 and ω-1, but when forcing the reaction with dicarboxylic acids, α- and β-hydroxylations were detected. Reactions with the α-ionone and α-damascone norisoprenoids produced major oxygenations at the cyclohexene and at the vinylic aliphatic chain, respectively. Taken together, PabUPO-II shares structural and functional similarities with both long and short UPOs, opening avenues for future engineering endeavors.