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Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain: a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biokemisk farmakologi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biokemisk farmakologi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biokemisk farmakologi)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biokemisk farmakologi)
2011 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 506, no 2, p. 216-222Article in journal (Refereed) Published
Abstract [en]

5,8-Linoleate diol synthase (5,8-LDS) of Aspergillus fumigatus was cloned, expressed, and compared with 7,8-LDS of the Take-all fungus. Replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8R-dioxygenase (8-DOX) and hydroperoxide isomerase activities, respectively. The predicted α-helices of LDS were aligned with α-helices of cyclooxygenase-1 (COX-1) to identify the 8-DOX domains. N-terminal expression constructs of 5,8- and 7,8-LDS (674 of 1079, and 673 of 1165 residues), containing one additional α-helix compared to cyclooxygenase-1, yielded prominent 8R-DOX activities with apparently unchanged or slightly lower substrate affinities, respectively. Val-328 of 5,8-LDS did not influence the position of oxygenation in contrast to the homologous residues Val-349 of COX-1 and Leu-384 of 10R-dioxygenase. We conclude that ∼675 amino acids are sufficient to support 8-DOX activity.

Place, publisher, year, edition, pages
2011. Vol. 506, no 2, p. 216-222
Keywords [en]
Cytochrome P450, fusion protein, heme-dependent peroxidase, hydroperoxide isomerase, LC-MS/MS, oxylipins
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemical Pharmacology
Identifiers
URN: urn:nbn:se:uu:diva-142985DOI: 10.1016/j.abb.2010.11.022ISI: 000286961600014PubMedID: 21130068OAI: oai:DiVA.org:uu-142985DiVA, id: diva2:388865
Available from: 2011-01-18 Created: 2011-01-18 Last updated: 2017-12-11Bibliographically 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. p. 68
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 135
Keywords
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
Identifiers
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)
Opponent
Supervisors
Available from: 2011-02-10 Created: 2011-01-19 Last updated: 2018-01-12Bibliographically approved
2. 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. p. 67
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 176
Keywords
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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