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Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical Pharmacology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical Pharmacology)
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 48, 34707-34718 p.Article in journal (Refereed) Published
Abstract [en]

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z- hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

Place, publisher, year, edition, pages
2007. Vol. 282, no 48, 34707-34718 p.
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-15043DOI: 10.1074/jbc.M705366200ISI: 000251155200013PubMedID: 17906293OAI: oai:DiVA.org:uu-15043DiVA: diva2:42814
Available from: 2008-02-04 Created: 2008-02-04 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Catalytic and Structural Properties of Heme-containing Fatty Acid Dioxygenases: Similarities of Fungal Dioxygenases and Cyclooxygenases
Open this publication in new window or tab >>Catalytic and Structural Properties of Heme-containing Fatty Acid Dioxygenases: Similarities of Fungal Dioxygenases and Cyclooxygenases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties.

The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site.

Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE.

10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8.

LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity.

LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 109
Keyword
dioxygenase, prostaglandin H synthase, Gaeumannomyces graminis, Aspergilli, hydroperoxide isomerase, linoleate diol synthase, cytochrome P450, oxylipin
National Category
Pharmacology and Toxicology
Research subject
Pharmaceutical Pharmacology
Identifiers
urn:nbn:se:uu:diva-108770 (URN)978-91-554-7624-3 (ISBN)
Public defence
2009-11-20, BMC, B42, Husargatan 3, 75123 Uppsala, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-10-28 Created: 2009-09-29 Last updated: 2011-05-11Bibliographically approved
2. 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 135
Keyword
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: 2011-03-11Bibliographically approved

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Garscha, UlrikeJernerén, FredrikOliw, Ernst H.

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