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Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biokemisk farmakologi)
Karolinska institutet.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Theoretical Chemistry.ORCID iD: 0000-0001-7567-8295
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2012 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1388-1918, Vol. 1821, no 12, 1508-1517 p.Article in journal (Refereed) Published
Abstract [en]

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O2. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n-6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n-6 and n-9 fatty acids and by analysis of D-KIE. COX-1 oxidized C20 and C18 fatty acids in the following order of rates: 20:2n-6 > 20:1n-6 > 20:3n-9 > 20:1n-9 and 18:3n-3 ≥ 18:2n-6 > 18:1n-6. 18:2n-6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n-6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n-6, but the 9E,12Z isomer was only subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-2H]18:2n-6 with similar D-KIE (~53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n-6 by COX-1 and COX-2 took place with a D-KIE of 3-5 as probed by incubations of [11,11-2H2]- and [11S-2H]18:2n-6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n-6 by COX-1 and 18:1n-9 by 8R-DOX were both accompanied by large D-KIE (>20).

Place, publisher, year, edition, pages
2012. Vol. 1821, no 12, 1508-1517 p.
Keyword [en]
Animal heme peroxidase, Chiral phase HPLC, Fatty acid oxygenation, Kinetic isotope effect, Mass spectrometry, Oxygenation mechanism
National Category
Biochemistry and Molecular Biology
Research subject
Biochemical Pharmacology
URN: urn:nbn:se:uu:diva-181786DOI: 10.1016/j.bbalip.2012.09.001ISI: 000310100900007OAI: oai:DiVA.org:uu-181786DiVA: diva2:557645
Knut and Alice Wallenberg Foundation, KAW 2004.0123Swedish Research Council, 06523
Available from: 2012-09-28 Created: 2012-09-28 Last updated: 2015-01-08Bibliographically 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

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