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Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical Pharmacology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical Pharmacology)
2007 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 367, no 2, 238-246 p.Article in journal (Refereed) Published
Abstract [en]

8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-2H]8-HODE and [10-2H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess–Martin periodinane followed by reduction to hydroxy fatty acids with NaB2H4. The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-2H2]5,8- and [8,11-2H2]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC–MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.

Place, publisher, year, edition, pages
2007. Vol. 367, no 2, 238-246 p.
Keyword [en]
Aspergillus, Chiralcel OB, 5, 8-DiHODE, 8, 11-DiHODE, Normal phase HPLC, Reprosil chiral NR, Ion trap mass spectrometry
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-15041DOI: 10.1016/j.ab.2007.04.045ISI: 000248198500012PubMedID: 17553451OAI: oai:DiVA.org:uu-15041DiVA: diva2:42812
Available from: 2008-02-04 Created: 2008-02-04 Last updated: 2011-01-28Bibliographically approved
In thesis
1. Catalytic and Structural Properties of Heme-containing Fatty Acid Dioxygenases: Similarities of Fungal Dioxygenases and Cyclooxygenases
Open this publication in new window or tab >>Catalytic and Structural Properties of Heme-containing Fatty Acid Dioxygenases: Similarities of Fungal Dioxygenases and Cyclooxygenases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties.

The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site.

Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE.

10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8.

LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity.

LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 58 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 109
dioxygenase, prostaglandin H synthase, Gaeumannomyces graminis, Aspergilli, hydroperoxide isomerase, linoleate diol synthase, cytochrome P450, oxylipin
National Category
Pharmacology and Toxicology
Research subject
Pharmaceutical Pharmacology
urn:nbn:se:uu:diva-108770 (URN)978-91-554-7624-3 (ISBN)
Public defence
2009-11-20, BMC, B42, Husargatan 3, 75123 Uppsala, Uppsala, 13:15 (English)
Available from: 2009-10-28 Created: 2009-09-29 Last updated: 2011-05-11Bibliographically approved

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