uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Gene deletion of 7,8-linoleate diol synthase of the rice blast fungus: studies on pathogenicity, stereochemistry, and oxygenation mechanisms
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Show others and affiliations
2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 8, 5308-5316 p.Article in journal (Refereed) Published
Abstract [en]

Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Delta7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Delta7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Delta mac1 sum1-99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.

Place, publisher, year, edition, pages
2010. Vol. 285, no 8, 5308-5316 p.
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-122730DOI: 10.1074/jbc.M109.062810ISI: 000275327200024PubMedID: 20023302OAI: oai:DiVA.org:uu-122730DiVA: diva2:311213
Available from: 2010-04-20 Created: 2010-04-16 Last updated: 2011-03-11Bibliographically approved
In thesis
1. Novel Fatty Acid Dioxygenases of Human and Plant Pathogenic Fungi: Studies by Gene Deletion and Expression
Open this publication in new window or tab >>Novel Fatty Acid Dioxygenases of Human and Plant Pathogenic Fungi: Studies by Gene Deletion and Expression
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The dioxygenase-cytochrome P450 fusion proteins (DOX-CYP) comprise a heme-containing enzyme family that shares structural and catalytic properties with mammalian prostaglandin H (PGH) synthases. 7,8-Linoleate diol synthase (7,8-LDS) of Gaeumannomyces graminis was first characterized, and DOX-CYP enzymes are of mechanistic and biological interest. The growing number of fungal genome sequences has revealed DOX-CYP homologues in medically and economically important species. The aim of this thesis was to identify novel members of the DOX-CYP fusion protein family.

The devastating rice pathogen Magnaporthe oryzae contains two DOX-CYP genes. The fungus synthesizes 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by dioxygenation of linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8R-HPODE), and subsequent isomerisation to the diol. 7,8-LDS of M. oryzae was identified by gene deletion, but the infection and reproduction processes of the Δ7,8-LDS strain were not altered. A mutant with constitutive protein kinase A activity profoundly changed the oxygenation profile, possibly due to post-translational modification.

The human pathogens Aspergillus fumigatus and A. clavatus contain three DOX-CYP, designated psi producing oxygenase A (ppoA), ppoB, and ppoC, and form three oxylipins: 5S,8R-DiHODE, 8R,11S-DiHODE, and 10R-hydroxyoctadecadienoic acid.  PpoA was identified as 5,8-LDS, and ppoC as 10R-DOX. The 8,11-linoleate hydroperoxide isomerase activity was reduced by two imidazole-containing P450 inhibitors, miconazole and 1-benzylimidazole. PpoB could not be linked to the biosynthesis of 8,11-DiHODE for the following reasons: First, the 8,11-hydroperoxide isomerase activity was retained in A. fumigatus ΔppoB strains. Second, the P450 domain of the deduced ppoB of A. clavatus lacks a heme-thiolate cysteine ligand, presumably essential for hydroperoxide isomerase activity.

Linoleate 9R-DOX activities of Aspergillus terreus and Lasiodiplodia theobromae were discovered. 9R-HPODE was further converted into unstable allene oxides, as judged by the accumulation of their hydrolysis products, α- and γ-ketols. These allene oxide synthase activities were specific for 9R-hydroperoxides. The 9R-DOX and AOS were found to have unique characteristics.

In conclusion, novel DOX-CYP enzymes were identified in human and plant pathogenic fungi. These enzymes might be involved in biological processes, and show interesting catalytic similarities to human PGH synthase and thromboxane synthase (CYP5A).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 68 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 135
aspergilli, dioxygenase, oxygenase, Magnaporthe oryzae, Gaeumannomyces graminis, Lasiodiplodia theobromae, jasmonic acid, linoleate diol synthase, cyclooxygenase, prostaglandin H synthase, cytochrome P450, oxylipin, hydroperoxide isomerase, allene oxide synthase
National Category
Pharmacology and Toxicology
Research subject
Biochemical Pharmacology
urn:nbn:se:uu:diva-143065 (URN)978-91-554-7987-9 (ISBN)
Public defence
2011-03-04, B22, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2011-02-10 Created: 2011-01-19 Last updated: 2011-03-11Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed
By organisation
Department of Pharmaceutical Biosciences
In the same journal
Journal of Biological Chemistry
Pharmaceutical Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 212 hits
ReferencesLink to record
Permanent link

Direct link