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Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450: Mechanisms of Oxylipin Biosynthesis in Pathogenic Fungi
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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 [en]
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: urn:nbn:se:uu:diva-206199ISBN: 978-91-554-8739-3 (print)OAI: oai:DiVA.org:uu-206199DiVA: diva2:644057
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
List of papers
1. Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain: a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
Open this publication in new window or tab >>Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain: a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
2011 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 506, no 2, 216-222 p.Article 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.

Keyword
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:nbn:se:uu:diva-142985 (URN)10.1016/j.abb.2010.11.022 (DOI)000286961600014 ()21130068 (PubMedID)
Available from: 2011-01-18 Created: 2011-01-18 Last updated: 2017-12-11Bibliographically approved
2. 7,8- and 5,8-linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues.
Open this publication in new window or tab >>7,8- and 5,8-linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues.
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. 

National Category
Other Natural Sciences Biochemistry and Molecular Biology
Research subject
Pharmaceutical Biochemistry; Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-206083 (URN)10.1016/j.abb.2013.09.010 (DOI)000326136000010 ()
Available from: 2013-08-29 Created: 2013-08-27 Last updated: 2017-12-06Bibliographically approved
3. Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs
Open this publication in new window or tab >>Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs
2012 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, 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).

Keyword
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
Identifiers
urn:nbn:se:uu:diva-181786 (URN)10.1016/j.bbalip.2012.09.001 (DOI)000310100900007 ()
Funder
Knut and Alice Wallenberg Foundation, KAW 2004.0123Swedish Research Council, 06523
Available from: 2012-09-28 Created: 2012-09-28 Last updated: 2017-12-07Bibliographically approved
4. Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus
Open this publication in new window or tab >>Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus
2010 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 495, no 1, 67-73 p.Article in journal (Refereed) Published
Abstract [en]

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-2H]18:2n-6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (alpha-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (gamma-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. alpha-Linolenic acid and 20:2n-6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but alpha- and gamma-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.

Keyword
Catalase, Cytochrome P450, 9R-HpODE, Heme peroxidase, Jasmonic acid, Oxygenation mechanism, Potato stolons
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-122731 (URN)10.1016/j.abb.2009.12.022 (DOI)000275137000011 ()20043865 (PubMedID)
Available from: 2010-04-20 Created: 2010-04-16 Last updated: 2017-12-12Bibliographically approved
5. Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase
Open this publication in new window or tab >>Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-200688 (URN)10.1074/jbc.M113.458257 (DOI)000317915500047 ()
Available from: 2013-06-03 Created: 2013-06-03 Last updated: 2017-12-06Bibliographically approved
6. Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum
Open this publication in new window or tab >>Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum
2013 (English)In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 54, no 12, 3417-3480 p.Article in journal (Refereed) Published
Abstract [en]

Fusarium oxysporum is a devastating plant pathogen that oxidizes C-18 fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e. g., FOXB_01332. Recombinant FOXB_01332 oxidized 18:2n-6 to 9S-hydroperoxy-10(E), 12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9S(10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (alpha- and gamma-ketols). The enzyme was therefore designated 9S-DOX-AOS. The 9S-DOX activity oxidized C-18 and C-20 fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB_01332 has acidic and alcoholic residues, Glu(946)-Val-Leu-Ser(949), at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB_01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. FOXB_01332 is the first linoleate 9-DOX with homology to animal heme peroxidases and the first 9-DOX-AOS fusion protein.

National Category
Biochemistry and Molecular Biology Other Natural Sciences
Research subject
Pharmaceutical Biochemistry; Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-206088 (URN)10.1194/jlr.M044347 (DOI)000330534900023 ()
Available from: 2013-08-29 Created: 2013-08-27 Last updated: 2017-12-06Bibliographically approved

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