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Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 16, 11459-11469 p.Article in journal (Refereed) Published
Abstract [en]

Aspergilli oxidize C-18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E), 12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R), 10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn(964) may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn(878) in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln(881) markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln(881) with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.

Place, publisher, year, edition, pages
2013. Vol. 288, no 16, 11459-11469 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-200688DOI: 10.1074/jbc.M113.458257ISI: 000317915500047OAI: oai:DiVA.org:uu-200688DiVA: diva2:624743
Available from: 2013-06-03 Created: 2013-06-03 Last updated: 2017-12-06Bibliographically 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 176
Keyword
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
Identifiers
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)
Opponent
Supervisors
Available from: 2013-09-27 Created: 2013-08-29 Last updated: 2014-01-23

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Hoffmann, IngaOliw, Ernst H.

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