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Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450

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Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 176 Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450 Mechanisms of Oxylipin Biosynthesis in Pathogenic Fungi INGA HOFFMANN ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-554-8739-3 UPPSALA urn:nbn:se:uu:diva-206199 2013 Dissertation presented at Uppsala University to be publicly examined in B21, BMC, Husargatan 3, Uppsala, Friday, October 18, 2013 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Abstract Hoffmann, I. 2013. Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450: Mechanisms of Oxylipin Biosynthesis in Pathogenic Fungi. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 176. 67 pp. Uppsala. ISBN 978-91-554-8739-3. 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. Keywords: Fusion protein, Linoleate diol synthase, Allene oxide synthase, Cyclooxygenase, Oxygenase, HPLC, Mass spectrometry, Hydroperoxide isomerase, Aspergillus, Fusarium oxysporum Inga Hoffmann, Uppsala University, Department of Pharmaceutical Biosciences, Box 591, SE-751 24 Uppsala, Sweden. © Inga Hoffmann 2013 ISSN 1651-6192 ISBN 978-91-554-8739-3 urn:nbn:se:uu:diva-206199 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-206199) List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Hoffmann, I., Jernerén, F., Garscha, U., Oliw, E.H. (2011) Ex- pression of 5,8-LDS of Aspergillus fumigatus and its dioxyge- nase domain. A comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase. Archives of Biochemistry and Biophysics, 506:216–22 II Hoffmann, I., Oliw, E.H. (2013) 7,8- And 5,8-linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues. Submitted manuscript III Hoffmann, I., Hamberg, M., Lindh, R., Oliw, E.H. (2012) Novel insights into cyclooxygenases, linoleate diol synthases, and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs. Biochimica et Biophysica Acta – Molecular and Cell Biology of Lipids, 1821:1508-17 IV Jernerén, F., Hoffmann, I., Oliw, E.H. (2010) Linoleate 9R- dioxygenase and allene oxide synthase activities of Aspergillus terreus. Archives of Biochemistry and Biophysics, 495:67–73 V Hoffmann, I., Jernerén, F., Oliw, E.H. (2013) Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase. The Journal of Biological Chemistry, 288:11459-69 VI Hoffmann, I., Oliw, E.H. (2013) Discovery of a Linoleate 9S- Dioxygenase and an Allene Oxide Synthase in a Fusion Protein of Fusarium oxysporum. Submitted manuscript Reprints were made with permission from Elsevier and the American Society for Biochemistry and Molecular Biology. Additional Papers • Jernerén, F., Garscha, U., Hoffmann, I., Hamberg, M., Oliw, E.H. (2010) Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Asper- gillus clavatus. Biochimica et Biophysica Acta – Molecular and Cell Biology of Lipids, 1801:503-7 • Oliw, E.H., Jernerén, F., Hoffmann, I., Sahlin, M., Garscha, U. (2011) Manganese lipoxygenase oxidizes bis-allylic hydroper- oxides and octadecenoic acids by different mechanisms. Bio- chimica et Biophysica Acta – Molecular and Cell Biology of Li- pids, 1811:138-47 • Oliw, E.H., Wennman, A., Hoffmann, I., Garscha, U., Hamberg, M., Jernerén, F. (2011) Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases. Journal of Lipid Research, 52:1995-2004 Contents Introduction ..................................................................................................... 9 Oxylipins .................................................................................................... 9 Mammalian oxylipins – Eicosanoids ..................................................... 9 Plant oxylipins ..................................................................................... 11 Fungal oxylipins .................................................................................. 12 Fatty acid oxygenases ............................................................................... 13 Fatty acid monooxygenases – Cytochrome P450 ................................ 14 Fatty acid dioxygenases ....................................................................... 16 Pathogenic fungi ....................................................................................... 22 Aims .............................................................................................................. 24 Comments on methodology .......................................................................... 25 Recombinant expression .......................................................................... 25 Expression in insect cells ..................................................................... 25 Expression in E. coli ............................................................................ 26 Analysis of protein expression ................................................................. 26 Site-directed mutagenesis ......................................................................... 27 Enzyme activity assay .............................................................................. 27 Analysis of fatty acid metabolites ............................................................ 27 High performance liquid chromatography ........................................... 27 Mass spectrometry ............................................................................... 28 Kinetic isotope effects .............................................................................. 29 Bioinformatics .......................................................................................... 30 Results ........................................................................................................... 31 Dioxygenase activities of LDS (paper I) ................................................. 31 Hydroperoxide isomerase activities of LDS (paper II and V) ................. 32 Novel insights into the reaction mechanisms of COX, 8R-DOX, and LOX (paper III) ........................................................................................ 34 Kinetic isotope effects of COX, 8R-DOX, 9-DOX, and LOX (paper III, V, and VI) ............................................................................................ 37 Oxylipin profile of A. terreus (paper IV) ................................................. 38 Discovery of the first fungal AOS (paper V) ........................................... 39 Crucial amide residue for AOS activity of A. terreus (paper V) .............. 40 10-DOX and 5,8-LDS of A. terreus (paper V) ......................................... 40 Discovery of a 9S-DOX-AOS fusion enzyme of F. oxysporum (paper VI) ............................................................................................................. 41 Discussion ..................................................................................................... 42 LDS are fusion proteins ............................................................................ 42 DOX activities ....................................................................................
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