title: The fatty acid 8,11-diol synthase of Aspergillus fumigatus is inhibited by imidazole derivatives and unrelated to PpoB.
abstract: (8R)-Hydroperoxy-(9Z,12Z)-octadecadienoic acid (8-HPODE) is formed by aspergilli as an intermediate in biosynthesis of oxylipins with effects on sporulation. 8-HPODE is transformed by separate diol synthases to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxy-(9Z,12Z)-octadecadienoic acids (5,8- and 8,11-DiHODE). The former is formed by the cytochrome P450 (P450) domain of 5,8-linoleate diol synthase (5,8-LDS or PpoA). Our aim was to characterize the 8,11-diol synthase of Aspergillus fumigatus, which is prominent in many strains. The 8,11-diol synthase was soluble and had a larger molecular size (>100 kDa) than most P450. Miconazole, ketoconazole, and 1-benzylimidazole, classical inhibitors of P450, reduced the biosynthesis of 8,11-DiHODE from 8-HPODE (apparent IC(50) values ~0.8, ~5, and ~0.6 μM, respectively), but did not inhibit the biosynthesis of 5,8-DiHODE. Analysis of hydroperoxides of regioisomeric C(18) and C(20) fatty acids showed that the 8,11-diol synthase was specific for certain hydroperoxides with R configuration. The suprafacial hydrogen abstraction and oxygen insertion at C-11 of 8-HPODE was associated with a small deuterium kinetic isotope effect ((H)                         k                         (cat)/(D)                         k                         (cat) ~1.5), consistent with P450-catalyzed oxidation. The genome of A. fumigatus contains over 70 P450 sequences. The reaction mechanism, size, and solubility of 8,11-diol synthase pointed to PpoB, a homologue of 5,8-LDS, as a possible candidate of this activity. Gene deletion of ppoB of A. fumigatus strains AF:∆ku80 and J272 did not inhibit biosynthesis of 8,11-DiHODE and recombinant PpoB appeared to lack diol synthase activity. We conclude that 8,11-DiHODE is formed from 8-HPODE by a soluble and substrate-specific 8,11-diol synthase with catalytic characteristics of class III P450.
authors.affiliation: Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, 75124, Uppsala, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Jernerén
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology*; D002853:Chromatography, Liquid; D005656:Fungal Proteins / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology*; D007654:Ketoconazole / Q000494:pharmacology; D008825:Miconazole / Q000494:pharmacology; D010105:Oxygenases / Q000378:metabolism*; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry
chemical_list: D005656:Fungal Proteins; D007093:Imidazoles; C017062:1-benzylimidazole; D008825:Miconazole; D010105:Oxygenases; C122014:linoleate diol synthase; D007654:Ketoconazole