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Pichia expression of and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase
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)
2008 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 373, no 4, 579-583 p.Article in journal (Refereed) Published
Abstract [en]

Linoleate diol synthases (LDS) are homologous 8(R)-dioxygenases with hydroperoxide isomerase activities, expressed in fungal pathogens of humanitarian importance. We report for the first time expression and site-directed mutagenesis of LDS. 7,8-LDS of the take-all fungus, expressed in Pichia pastoris, oxygenated 18:2n − 6 to 8(R)-hydroperoxylinoleic acid, which was unexpectedly isomerized to 5,8(R)-dihydroxylinoleic acid (60% 5S) and to 8(R),13-dihydroxyoctadeca-9(E),11(E)-dienoic acid. The latter was likely formed via hydrolysis of an unstable intermediate, 8(R),9(S)-epoxyoctadeca-10(E),12(Z)-dienoic acid. A tyrosyl radical is formed during 7,8-LDS catalysis, and Tyr376 is the sequence homolog to Tyr385 of cyclooxygenase-1. Tyr376Phe retained hydroperoxide isomerase activity but lacked 8(R)-dioxygenase activity. The putative proximal heme ligand His379 and the N-glycosylation site at Asn216 appeared to be critical for 8(R)-dioxygenase activity, as His379Gln and Asn216Gln were inactive. Treatment with α-mannosidase to shorten N- and O-linked mannosides inhibited the hydroperoxide isomerase but not the 8(R)-dioxygenase. Our results suggest that post-translational modifications may influence the oxidation mechanism of 7,8-LDS.

Place, publisher, year, edition, pages
2008. Vol. 373, no 4, 579-583 p.
Keyword [en]
Aspergillus nidulans, Dioxygenase, Gaeumannomyces graminis, Hemoprotein, Hydroperoxide isomerase, HPLC, α-Mannosidase, Mass-spectrometry, Myeloperoxidase
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-86644DOI: 10.1016/j.bbrc.2008.06.060ISI: 000258208500023PubMedID: 18586008OAI: oai:DiVA.org:uu-86644DiVA: diva2:126743
Available from: 2008-11-27 Created: 2008-11-20 Last updated: 2010-06-07Bibliographically 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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