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 Asn, Arg, and Arg, are catalytic importance and belong to the catalytic groove. Phe and Gln 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.