Molecular characterization of Penicillium oxalicum 6R,8R-linoleate diol synthase with new regiospecificity

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn⁸⁸⁶, Arg⁷⁰⁷, and Arg⁹³⁴, are catalytic importance and belong to the catalytic groove. Phe⁷⁹⁴ and Gln⁸⁸⁹ were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.