The Fatty Acid 8,11-Diol Synthase of Aspergillus fumigatus is Inhibited by Imidazole Derivatives and Unrelated to PpoB
2012 (English)In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 47, no 7, 707-717 p.Article in journal (Refereed) Published
(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 IC50 values similar to 0.8, similar to 5, and similar to 0.6 mu M, 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) similar to 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:Delta 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.
Place, publisher, year, edition, pages
2012. Vol. 47, no 7, 707-717 p.
Gene deletion, Sporulation, Animal heme peroxidase, LC-MS/MS, Insect cells, Protein expression
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-179936DOI: 10.1007/s11745-012-3673-2ISI: 000306789400007OAI: oai:DiVA.org:uu-179936DiVA: diva2:547121
FunderKnut and Alice Wallenberg Foundation, KAW 2004.0123