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

Direct link
7,8- and 5,8-linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2013 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 539, no 1, 87-91 p.Article in journal (Other academic) Published
Abstract [en]

Linoleate diol synthases (LDS) are fungal dioxygenase-cytochrome P450 fusion enzymes. They oxidize 18:2n-6 sequentially to 8R-hydroperoxylinoleic acid (8R-HPODE) and 7S,8S- or 5S,8R-dihydroxylinoleic acids (DiHODE) by intramolecular oxygen transfer. The P450 domains contain a conserved sequence, Ala-Asn-Gln-Xaa-Gln, presumably located in the I-helices. The Asn938Leu replacement of 7,8-LDS of Gaeumannomyces graminis virtually abolished and the Asn938Asp and Asn938Gln replacements reduced the hydroperoxide isomerase activity. Gln941Leu and Gln941Glu substitutions had little effects. Replacements of the homologous Asn(887) and Gln(890) residues of 5,8-LDS of Aspergillus fumigatus yielded the opposite results. Asn887Leu and Asn887Gln of 5,8-LDS retained 5,8-DiHODE as the main metabolite with an increased formation of 6,8- and 8,11-DiHODE, whereas Gln890Leu almost abolished the 5,8-LDS activity. Replacement of Gln(890) with Glu also retained 5,8-DiHODE as the main product, but shifted oxygenation from C-5 to C-7 and C-11 and to formation of epoxyalcohols by homolytic scission of 8R-HPODE. P450 hydroxylases usually contain an "acid-alcohol" pair in the I-helices for the heterolytic scission of O-2 and formation of compound I (Por(+.) Fe(IV)=0) and water. The function of the acid-alcohol pair appears to be replaced by two different amide residues, Asn(938) of 7,8-LDS and Gln(890) of 5,8-LDS, for heterolysis of 8R-HPODE to generate compound I. 

Place, publisher, year, edition, pages
2013. Vol. 539, no 1, 87-91 p.
National Category
Other Natural Sciences Biochemistry and Molecular Biology
Research subject
Pharmaceutical Biochemistry; Pharmaceutical Science
URN: urn:nbn:se:uu:diva-206083DOI: 10.1016/j.abb.2013.09.010ISI: 000326136000010OAI: oai:DiVA.org:uu-206083DiVA: diva2:644006
Available from: 2013-08-29 Created: 2013-08-27 Last updated: 2014-01-23Bibliographically approved
In thesis
1. Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450: Mechanisms of Oxylipin Biosynthesis in Pathogenic Fungi
Open this publication in new window or tab >>Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450: Mechanisms of Oxylipin Biosynthesis in Pathogenic Fungi
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Dioxygenase-cytochrome P450 (DOX-CYP) fusion enzymes are present in diverse human and plant pathogenic fungi. They oxygenate fatty acids to lipid mediators which have regula­tory functions in fungal development and toxin production. These enzymes catalyze the for­mation of fatty acid hy­droperoxides which are subsequently converted by the P450 activities or reduced to the corresponding alcohols. The N-terminal DOX domains show catalytic and structural homology to mammalian cyclooxygenases, which belong to the most thoroughly studied human enzymes.

7,8-Linoleate diol synthase (LDS) of the plant pathogenic fungus Gaeumannomyces graminis was the first characterized member of the DOX-CYP fusion enzyme family. It catalyzes the conversion of linoleic acid to 8R-hydroperoxylinoleic acid (HPODE) and subse­quently to 7S,8S-dihy­droxylinoleic acid by its DOX and P450 domains, respectively. By now, several enzymes with homology to 7,8-LDS have been identified in im­portant fungi, e.g., psi fac­tor-producing oxygenase (ppo)A, ppoB, and ppoC, of Aspergillus nidulans and A. fumigatus.

By cloning and recombinant expression, ppoA of A. fumigatus was identi­fied as 5,8-LDS. Partial expression of the 8R-DOX domains of 5,8-LDS of A. fumigatus and 7,8-LDS of G. graminis yielded active protein which demonstrates that the DOX activities of LDS are independent of their P450 domains. The latter domains were shown to contain a conserved motif with catalytically important amide residues. As judged by site-directed mutagene­sis studies, 5,8- and 7,8-LDS seem to facilitate heterolytic cleavage of the oxygen-oxygen bond of 8R-HPODE by aid of a glutamine and an asparagine residue, respectively.

Cloning and expression of putative DOX-CYP fusion proteins of A. terreus and Fusarium oxysporum led to the discovery of novel enzyme activities, e.g., linoleate 9S-DOX and two allene oxide synthases (AOS), specific for 9R- and 9S-HPODE, respectively. The fungal AOS are present in the P450 domains of two DOX-CYP fusion enzymes and show higher se­quence homology to LDS than to plant AOS and constitute therefore a novel class of AOS.

In summary, this thesis describes the discovery of novel fatty acid oxy­genases of human and plant pathogenic fungi and the characterization of their reaction mechanisms.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 67 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 176
Fusion protein, Linoleate diol synthase, Allene oxide synthase, Cyclooxygenase, Oxygenase, HPLC, Mass spectrometry, Hydroperoxide isomerase, Aspergillus, Fusarium oxysporum
National Category
Biochemistry and Molecular Biology Other Natural Sciences
Research subject
Pharmaceutical Biochemistry; Pharmaceutical Pharmacology; Pharmaceutical Science
urn:nbn:se:uu:diva-206199 (URN)978-91-554-8739-3 (ISBN)
Public defence
2013-10-18, B21, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2013-09-27 Created: 2013-08-29 Last updated: 2014-01-23

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Hoffmann, IngaOliw, Ernst H.
By organisation
Department of Pharmaceutical Biosciences
In the same journal
Archives of Biochemistry and Biophysics
Other Natural SciencesBiochemistry and Molecular Biology

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: 183 hits
ReferencesLink to record
Permanent link

Direct link