Title: Identification of Dioxygenases Required for Aspergillus Development
Abstract: 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. 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. The Aspergilli constitute a family of ascomycete fungi (1Wilson D.M. Mubatanhema W. Jurjevic Z. Adv. Exp. Med. Biol. 2002; 504: 3-17Crossref PubMed Scopus (89) Google Scholar, 2Jones M.G. Microbiology. 2007; 153: 1-6Crossref PubMed Scopus (42) Google Scholar). Several species are important human allergens, opportunistic pathogens, and producers of mycotoxins. Their spores are ubiquitous in the environment. Immunocompromised patients are particularly vulnerable to infections by Aspergillus fumigatus, causing farmer's lung disease and invasive aspergillosis (1Wilson D.M. Mubatanhema W. Jurjevic Z. Adv. Exp. Med. Biol. 2002; 504: 3-17Crossref PubMed Scopus (89) Google Scholar, 2Jones M.G. Microbiology. 2007; 153: 1-6Crossref PubMed Scopus (42) Google Scholar). Aspergilli are also plant pathogens and used as industrial microorganisms. Aspergillus nidulans is a model organism for studies of fungal biology (3Doonan J.H. J. Cell Sci. 1992; 103: 599-611PubMed Google Scholar). The genomes of nine Aspergillus sp. have now been fully or partly sequenced, which highlights their biological importance (2Jones M.G. Microbiology. 2007; 153: 1-6Crossref PubMed Scopus (42) Google Scholar). One set of molecules known to be critical in Aspergillus developmental processes are a series of oxygenated fatty acids originally termed as psi 5The abbreviations used are: psi, precocious sexual inducer; CP, chiral phase; DiHODE, dihydroxyoctadecadienoic acid; DOX, dioxygenase; HODE, hydroxyoctadecadienoic acid; HOME, hydroxyoctadecenoic acid; HPODE, hydroperoxyoctadecadienoic acid; KODE, ketooctadecadienoic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDS, linoleate diol synthase; NP, normal phase; MPO, myeloperoxidase; ODA, 10-oxy-8E-decenoic acid; Ppo, psi producing oxygenase; ppo, gene coding for Ppo; RP-HPLC, reversed phase-high pressure liquid chromatography; TPP, triphenylphosphine; TNM, tetranitromethane. 5The abbreviations used are: psi, precocious sexual inducer; CP, chiral phase; DiHODE, dihydroxyoctadecadienoic acid; DOX, dioxygenase; HODE, hydroxyoctadecadienoic acid; HOME, hydroxyoctadecenoic acid; HPODE, hydroperoxyoctadecadienoic acid; KODE, ketooctadecadienoic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDS, linoleate diol synthase; NP, normal phase; MPO, myeloperoxidase; ODA, 10-oxy-8E-decenoic acid; Ppo, psi producing oxygenase; ppo, gene coding for Ppo; RP-HPLC, reversed phase-high pressure liquid chromatography; TPP, triphenylphosphine; TNM, tetranitromethane. factors. Champe and co-workers (4Champe S.P. el-Zayat A.A. J. Bacteriol. 1989; 171: 3982-3988Crossref PubMed Google Scholar, 5Mazur P. Meyers H.V. Nakanishi K. El-Zayat A.A.E. Champe S.P. Tetrahedron Lett. 1990; 31: 3837-3840Crossref Scopus (52) Google Scholar) showed in 1989 that A. nidulans oxidized 18:2n-6 and 18:1n-9 to psi factors, e.g. (8R)-HODE, (5S,8R)-DiHODE, and (8R)-HOME, which were identified as inducers of precocious sexual sporulation. Oxidation of polyunsaturated fatty acids to biologically active metabolites was well established in mammals and plants at that time, but this appears to be the first report of hormone-like activities of fungal oxylipins. Oxidation of 18:2n-6 to (8R)-HODE and DiHODE was not restricted to A. nidulans. (8R)-HODE was originally discovered in Laetisaria arvalis (6Bowers W.S. Hoch H.C. Evans P.H. Katayama M. Science. 1986; 232: 105-106Crossref PubMed Scopus (55) Google Scholar, 7Brodowsky I.D. Oliw E.H. Biochim. Biophys. Acta. 1993; 1168: 68-72Crossref PubMed Scopus (39) Google Scholar). (8R)-HODE is also produced by other fungi, e.g. Gaeumannomyces graminis (the take-all fungus of wheat), Magnaporthe grisea (the rice blast fungus), Leptomitus lacteus (the sewage fungus), and Agaricus bisporus (the field mushroom) (8Brodowsky I.D. Oliw E.H. Biochim. Biophys. Acta. 1992; 1124: 59-65Crossref PubMed Scopus (55) Google Scholar, 9Fox S.R. Akpinar A. Prabhune A.A. Friend J. Ratledge C. Lipids. 2000; 35: 23-30Crossref PubMed Scopus (24) Google Scholar, 10Cristea M. Osbourn A.E. Oliw E.H. Lipids. 2003; 38: 1275-1280Crossref PubMed Scopus (25) Google Scholar, 11Wadman M.W. van Zadelhoff G. Hamberg M. Visser T. Veldink G.A. Vliegenthart J.F. Lipids. 2005; 40: 1163-1170Crossref PubMed Scopus (19) Google Scholar). G. graminis and M. grisea also form (7S,8S)-DiHODE and the field mushroom (8R,11S)-DiHODE (8Brodowsky I.D. Oliw E.H. Biochim. Biophys. Acta. 1992; 1124: 59-65Crossref PubMed Scopus (55) Google Scholar, 10Cristea M. Osbourn A.E. Oliw E.H. Lipids. 2003; 38: 1275-1280Crossref PubMed Scopus (25) Google Scholar, 11Wadman M.W. van Zadelhoff G. Hamberg M. Visser T. Veldink G.A. Vliegenthart J.F. Lipids. 2005; 40: 1163-1170Crossref PubMed Scopus (19) Google Scholar). The mechanism of biosynthesis of (8R)-HODE and (7S,8S)-DiHODE was determined in G. graminis (12Brodowsky I.D. Hamberg M. Oliw E.H. J. Biol. Chem. 1992; 267: 14738-14745Abstract Full Text PDF PubMed Google Scholar, 13Su C. Oliw E.H. J. Biol. Chem. 1996; 271: 14112-14118Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 14Su C. Sahlin M. Oliw E.H. J. Biol. Chem. 1998; 273: 20744-20751Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 18:2n-6 was oxidized to (8R)-HPODE by a heme-containing (8R)-DOX with hydroperoxide isomerase activity, 7,8-LDS. This enzyme abstracts the pro-S hydrogen at C-8 of 18:2n-6 and forms a carbon-centered radical, which reacts with O2 in an antaraficial way and forms (8R)-HPODE (15Hamberg M. Gerwick W.H. Åsén P.A. Lipids. 1992; 27: 487-493Crossref Scopus (42) Google Scholar). A tyrosyl radical can be detected by EPR in this process (14Su C. Sahlin M. Oliw E.H. J. Biol. Chem. 1998; 273: 20744-20751Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). (8R)-HPODE is isomerized to (7S,8S)-DiHODE by suprafacial hydrogen abstraction and oxygenation at C-7 (16Hamberg M. Zhang L.Y. Brodowsky I.D. Oliw E.H. Arch. Biochem. Biophys. 1994; 309: 77-80Crossref PubMed Scopus (43) Google Scholar), catalyzed by a ferryl intermediate (PPIX Fe4+ = O) (14Su C. Sahlin M. Oliw E.H. J. Biol. Chem. 1998; 273: 20744-20751Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 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Nature. 2005; 438: 1105-1115Crossref PubMed Scopus (1042) Google Scholar, 25Nierman W.C. Pain A. Anderson M.J. Wortman J.R. Kim H.S. Arroyo J. Berriman M. Abe K. Archer D.B. Bermejo C. Bennett J. Bowyer P. Chen D. Collins M. Coulsen R. Davies R. Dyer P.S. Farman M. Fedorova N. Fedorova N. Feldblyum T.V. Fischer R. Fosker N. Fraser A. Garcia J.L. Garcia M.J. Goble A. Goldman G.H. Gomi K. Griffith-Jones S. Gwilliam R. Haas B. Haas H. Harris D. Horiuchi H. Huang J. Humphray S. Jimenez J. Keller N. Khouri H. Kitamoto K. Kobayashi T. Konzack S. Kulkarni R. Kumagai T. Lafon A. Latge J.P. Li W. Lord A. Lu C. Majoros W.H. May G.S. Miller B.L. Mohamoud Y. Molina M. Monod M. Mouyna I. Mulligan S. Murphy L. O'Neil S. Paulsen I. Penalva M.A. Pertea M. Price C. Pritchard B.L. Quail M.A. Rabbinowitsch E. Rawlins N. Rajandream M.A. Reichard U. Renauld H. Robson G.D. Rodriguez de Cordoba S. Rodriguez-Pena J.M. Ronning C.M. Rutter S. Salzberg S.L. Sanchez M. Sanchez-Ferrero J.C. Saunders D. Seeger K. Squares R. Squares S. Takeuchi M. Tekaia F. Turner G. Vazquez de Aldana C.R. Weidman J. White O. Woodward J. Yu J.H. Fraser C. Galagan J.E. Asai K. Machida M. Hall N. Barrell B. Denning D.W. Nature. 2005; 438: 1151-1156Crossref PubMed Scopus (1079) Google Scholar). Keller and co-workers found with the aid of the 7,8-LDS sequence that both genomes contained three genes (ppoA, ppoB, and ppoC), which coded for putative fatty acid oxygenases of the MPO family with about 40% amino acid identity with 7,8-LDS (22Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Microbiology. 2005; 151: 1809-1821Crossref PubMed Scopus (135) Google Scholar). The exon-intron borders and the amino acid sequences of the gene transcripts could be deduced from sequence homology to 7,8-LDS, including homology to the presumed distal and proximal heme ligands of 7,8-LDS and the critical Tyr residue for catalysis. 6U. Garscha and E. H. Oliw, submitted for publication. 6U. Garscha and E. H. Oliw, submitted for publication. The deduced sequence of PpoA of A. nidulans was confirmed by cDNA analysis (23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Keller and co-workers (20Tsitsigiannis D.I. Keller N.P. Mol. Microbiol. 2006; 59: 882-892Crossref PubMed Scopus (126) Google Scholar, 21Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Eukaryot. Cell. 2004; 3: 1398-1411Crossref PubMed Scopus (99) Google Scholar, 22Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Microbiology. 2005; 151: 1809-1821Crossref PubMed Scopus (135) Google Scholar, 23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) reported that deletion of these genes affected the ratio of asexual spores (conidia) to sexual spores (ascopores), the biosynthesis of (8R)-HODE, and mycotoxin production in A. nidulans. In addition to the impact on the sporulation process, deletion of these genes also led to alterations in virulence on host seed (20Tsitsigiannis D.I. Keller N.P. Mol. Microbiol. 2006; 59: 882-892Crossref PubMed Scopus (126) Google Scholar). Deletion of ppoA reduced formation of (8R)-HODE and increased the ratio of conidia to ascospores, whereas forced expression of ppoA had the opposite effect (23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Most recently, deletion of ppoB also increased conidia formation, whereas deletion of ppoC decreased conidia formation (20Tsitsigiannis D.I. Keller N.P. Mol. Microbiol. 2006; 59: 882-892Crossref PubMed Scopus (126) Google Scholar, 21Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Eukaryot. Cell. 2004; 3: 1398-1411Crossref PubMed Scopus (99) Google Scholar). These results were recently extended to A. fumigatus. An initial study demonstrated that down-regulation of all three A. fumigatus ppo genes by RNA interference technology produced a hypervirulent strain (27Tsitsigiannis D.I. Bok J.W. Andes D. Nielsen K.F. Frisvad J.C. Keller N.P. Infect. Immun. 2005; 73: 4548-4559Crossref PubMed Scopus (98) Google Scholar). Further work showed that deletion of ppoC yielded a pleiotrophic phenotype with formation of aberrant conidia and increased virulence in a mouse model of aspergillosis. 7D. Chung, unpublished data. 7D. Chung, unpublished data. The biological effects of ppo gene loss in A. nidulans and A. fumigatus are summarized in Table 1.TABLE 1Biological effects of ppo gene loss in Aspergillus sp.CharacteristicsRef.A. nidulans mutants ΔppoAIncreased asexual spore, decreased sexual spore23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar ΔppoBIncreased asexual spore, decreased sexual spore23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google ScholarEnhanced virulenceaVirulence was assessed on host seed (peanut and maize). ΔppoCIncreased sexual spore, decreased asexual spore23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar ΔppoA,CIncreased sexual spore, decreased asexual spore21Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Eukaryot. Cell. 2004; 3: 1398-1411Crossref PubMed Scopus (99) Google Scholar, 22Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Microbiology. 2005; 151: 1809-1821Crossref PubMed Scopus (135) Google ScholarDecreased virulenceaVirulence was assessed on host seed (peanut and maize).A. fumigatus mutants ΔppoANone detected34de Boer H.J. Kool A. Broberg A. Mziray W.R. Hedberg I. Levenfors J.J. J. Ethnopharmacol. 2005; 96: 461-469Crossref PubMed Scopus (166) Google Scholar ΔppoBNone detectedFootnote 9 ΔppoCDecreased asexual spore, cell wall aberranciesFootnote 7Enhanced resistance to oxidative stressGermination defects, enhanced virulence attributesbVirulence was assessed in a murine model of aspergillosis. RNA interference ppoABCcThe RNA interference ppoABC strain was created by using RNA interference technology to knock down expression of all three ppo genes simultaneously. However, there is still residual gene expression of all three genes (27).Enhanced resistance to oxidative stress27Tsitsigiannis D.I. Bok J.W. Andes D. Nielsen K.F. Frisvad J.C. Keller N.P. Infect. Immun. 2005; 73: 4548-4559Crossref PubMed Scopus (98) Google ScholarEnhanced virulenceaVirulence was assessed on host seed (peanut and maize).a Virulence was assessed on host seed (peanut and maize).b Virulence was assessed in a murine model of aspergillosis.c The RNA interference ppoABC strain was created by using RNA interference technology to knock down expression of all three ppo genes simultaneously. However, there is still residual gene expression of all three genes (27Tsitsigiannis D.I. Bok J.W. Andes D. Nielsen K.F. Frisvad J.C. Keller N.P. Infect. Immun. 2005; 73: 4548-4559Crossref PubMed Scopus (98) Google Scholar). Open table in a new tab Studies of recombinant 7,8-LDS suggested that (5S,8R)-DiHODE could be formed by an enzyme of A. nidulans with a closely related oxygenation mechanism (26Coffa G. Schneider C. Brash A.R. Biochem. Biophys. Res. Commun. 2005; 338: 87-92Crossref PubMed Scopus (80) Google Scholar). 7,8-LDS expressed in insect cells had similar properties as the native enzyme. 7,8-LDS expressed in Pichia pastoris oxygenated 18:2n-6 to (8R)-HPODE and (5,8R)-DiHODE and transformed exogenous (8R)-HPODE to (5,8R)-DiHODE (26Coffa G. Schneider C. Brash A.R. Biochem. Biophys. Res. Commun. 2005; 338: 87-92Crossref PubMed Scopus (80) Google Scholar). Mycelia and cell-free preparations of A. nidulans were found to oxidize 18:2n-6 to (8R)-HPODE and (5S,8R)-DiHODE and transformed (8R)-HPODE to (5S,8R)-DiHODE. In addition, (10R)-HODE was formed as a major metabolite under certain conditions (26Coffa G. Schneider C. Brash A.R. Biochem. Biophys. Res. Commun. 2005; 338: 87-92Crossref PubMed Scopus (80) Google Scholar, 29Garscha U. Oliw E.H. Anal. Biochem. 2007; 367: 238-246Crossref PubMed Scopus (37) Google Scholar). Fungi have been known to produce 10-HODE with R or S absolute configuration. The shiitake mushroom, Lentinula edodes, and the field mushroom form (10S)-HPODE, which can be transformed to an aroma compound, 1-octen-3-ol, or reduced to (10S)-HODE (30Wurzenberger M. Grosch W. Biochim. Biophys. Acta. 1984; 794: 25-30Crossref Scopus (110) Google Scholar, 31Akakabe Y. Matsui K. Kajiwara T. Biosci. Biotechnol. Biochem. 2005; 69: 1539-1544Crossref PubMed Scopus (19) Google Scholar). (10R)-HODE is formed by Epichlöe typhina (32Koshino H. Togiya S. Yoshihara T. Sakamura S. Tetrahedron Lett. 1987; 28: 73-76Crossref Scopus (71) Google Scholar), and this stereoisomer also predominates in G. graminis and A. nidulans (26Coffa G. Schneider C. Brash A.R. Biochem. Biophys. Res. Commun. 2005; 338: 87-92Crossref PubMed Scopus (80) Google Scholar, 29Garscha U. Oliw E.H. Anal. Biochem. 2007; 367: 238-246Crossref PubMed Scopus (37) Google Scholar, 33Brodowsky I.D. Zhang L.Y. Oliw E.H. Hamberg M. Ann. N. Y. Acad. Sci. 1994; 744: 314-316Crossref PubMed Scopus (4) Google Scholar). The corresponding (10R)-hydroperoxide has not been identified, and little is known about the mechanism of biosynthesis of (10R)-HODE. The first aim of the present study was to examine the biosynthesis of oxylipins from 18:2n-6 by the human pathogen A. fumigatus. We next extended these studies to A. nidulans, as this has traditionally been used as a model organism (4Champe S.P. el-Zayat A.A. J. Bacteriol. 1989; 171: 3982-3988Crossref PubMed Google Scholar). We found that both species transformed 18:2n-6 to (8R)-HPODE/(8R)-HODE, (5S,8R)-DiHODE, and (10R)-HPODE/(10R)-HODE. In addition, (8R,11S)-DiHODE was formed by A. fumigatus. The second aim was to determine the mechanism of biosynthesis of the Aspergillus metabolites and their relation to (8R)-HPODE. The third goal was to determine the stereochemical relation between hydrogen abstraction and oxygenation using stereospecifically deuterated 18:2n-6 at C-5, C-8, and C-11. These studies were consistent with expression of two enzymes, 5,8-LDS and (10R)-DOX, in A. nidulans and A. fumigatus, and a third enzyme, 8,11-LDS, in A. fumigatus. Our final goal was to link each of the genes to each of these enzymes by gene targeting. We report that ppoA likely codes for 5,8-LDS and ppoC for (10R)-DOX. The corresponding genes appear to occur in virtually all Aspergillus sp. sequenced so far. 18:1n-9 (99%), 18:2n-6 (99%), 18:3n-3 (99%), 18:3n-6 (99%), and imidazole were from Merck. 18:2n-6 (94–96%) was from Carl Roth (Karlsruhe, Germany). [9,10,12,13-2H4]18:2n-6 (99%), 16:3n-3 (99%), 17:3n-3 (99%), 19:3n-3 (99%), 20:2n-6 (99%), 10-KODE (99%), and 10-ODA (99%) were obtained from Larodan (Malmö, Sweden). 16:1n-7 (99%), malt extract, and TNM were from Sigma. [(11S)-2H]18:2n-6 (>95%) and [(11R)-2H]18:2 (25%) were prepared as described (16Hamberg M. Zhang L.Y. Brodowsky I.D. Oliw E.H. Arch. Biochem. Biophys. 1994; 309: 77-80Crossref PubMed Scopus (43) Google Scholar), whereas [(8R)-2H]18:2n-6 and [(5S)-2H]18:2n-6 were synthesized as described in the Supplemental Material. The strains of Aspergillus sp. are summarized in Table 2 (cf. Refs. 20Tsitsigiannis D.I. Keller N.P. Mol. Microbiol. 2006; 59: 882-892Crossref PubMed Scopus (126) Google Scholar, 21Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Eukaryot. Cell. 2004; 3: 1398-1411Crossref PubMed Scopus (99) Google Scholar, 22Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Microbiology. 2005; 151: 1809-1821Crossref PubMed Scopus (135) Google Scholar, 23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). A. fumigatus Fres. will be referred to as A. fumigatus, and this strain was a kind gift of Dr. Levenfors (MASE Laboratories, Sveriges Lantbruksuniversitet, Uppsala, Sweden (34de Boer H.J. Kool A. Broberg A. Mziray W.R. Hedberg I. Levenfors J.J. J. Ethnopharmacol. 2005; 96: 461-469Crossref PubMed Scopus (166) Google Scholar)). Spores of A. fumigatus were isolated from mycelia on potato dextrose agar, harvested in 0.5% Bactopeptone (5 × 107 spores/ml), and kept at 4 °C. Solvents were HPLC grade from Merck and J. T. Baker Inc. Cartridges with C18 silica and silica (SepPak/C18 and SepPak, respectively) were from Waters. BW4AC was a kind gift from Wellcome Research Laboratories (Beckenham, UK), and stock solutions (10 mm) were made in ethanol. Zileuton was from Abbott. Paracetamol (acetaminophen) was obtained locally. (8R,11S)-DiHODE was obtained as described (11Wadman M.W. van Zadelhoff G. Hamberg M. Visser T. Veldink G.A. Vliegenthart J.F. Lipids. 2005; 40: 1163-1170Crossref PubMed Scopus (19) Google Scholar, 29Garscha U. Oliw E.H. Anal. Biochem. 2007; 367: 238-246Crossref PubMed Scopus (37) Google Scholar).TABLE 2Strains of Aspergillus sp. used in this studyGenotypeRef.Strains of A. fumigatusaThe A. fumigatus ppoA, ppoB, and ppoC genes were identified as described previously (27). Fres.Wild type34de Boer H.J. Kool A. Broberg A. Mziray W.R. Hedberg I. Levenfors J.J. J. Ethnopharmacol. 2005; 96: 461-469Crossref PubMed Scopus (166) Google Scholar AF293Wild type41Xue T. Nguyen C.K. Romans A. Kontoyiannis D.P. May G.S. Arch. Microbiol. 2004; 182: 346-353Crossref PubMed Scopus (57) Google Scholar TDWC1.13bThe ppoA and ppoB knock outs were prepared by homologous recombination in the pyrG1 auxotrophic strain AF293.1 using A. parasiticus pyrG both as replacement cassette and marker gene, details in Footnote 7.ΔppoA::A. parasiticus(A.p) pyrG; pyrG1Footnote 7 TDWC2.4bThe ppoA and ppoB knock outs were prepared by homologous recombination in the pyrG1 auxotrophic strain AF293.1 using A. parasiticus pyrG both as replacement cassette and marker gene, details in Footnote 7.ΔppoB::A. parasiticus(A.p) pyrG; pyrG1Footnote 7 TDWC4.17cThe ppoC knock out was prepared using the A. nidulans argB cassette and the pyrG1 and argB1 double auxotrophic strain AF293.6.7ΔppoC::A.n argB; argB1; pyrG1; ppoC::pyrGFootnote 7 TDWC10.5dTDWC10.5 was obtained by ectopic complementation with 5 kb of ppoC (containing 1 kb of its promoter7). All mutants were characterized by PCR and Southern hybridization.7Wild typeFootnote 7(ΔppoC::A. nidulans(A.n) argB; argB1; A.p pyrG; pyrG1)Strains of A. nidulans RDIT9.32veA, wild type23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar RDIT12.9methG1; ΔppoA::methG; veA23Tsitsigiannis D.I. Zarnowski R. Keller N.P. J. Biol. Chem. 2004; 279: 11344-11353Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar RDIT59.1pyroA4; ΔppoB::pyroA; veA22Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Microbiology. 2005; 151: 1809-1821Crossref PubMed Scopus (135) Google Scholar RDIT58.12ΔppoC::trpC; veA; trpC80121Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Eukaryot. Cell. 2004; 3: 1398-1411Crossref PubMed Scopus (99) Google Scholara The A. fumigatus ppoA, ppoB, and ppoC genes were identified as described previously (27Tsitsigiannis D.I. Bok J.W. Andes D. Nielsen K.F. Frisvad J.C. Keller N.P. Infect. Immun. 2005; 73: 4548-4559Crossref PubMed Scopus (98) Google Scholar).b The ppoA and ppoB knock outs were prepared by homologous recombination in the pyrG1 auxotrophic strain AF293.1 using A. parasiticus pyrG both as replacement cassette and marker gene, details in Footnote 7.c The ppoC knock out was prepared using the A. nidulans argB cassette and the pyrG1 and argB1 double auxotrophic strain AF293.6.7d TDWC10.5 was obtained by ectopic complementation with 5 kb of ppoC (containing 1 kb of its promoter7). All mutants were characterized by PCR and Southern hybridization.7 Open table in a new tab Fungal Growth—A. fumigatus and A. nidulans were grown in liquid media (1.5% malt extract) from spores or mycelia on agar in a rotary shaker (150 rpm) at 37 or 22 °C (dark or in laboratory light) for 3–10 days. Mycelia were harvested by filtration, washed with saline, and either used directly or blotted dry and ground to a fine powder in liquid nitrogen. A. fumigatus and A. nidulans were also grown in 9-cm plastic Petri dishes either in the dark or 50 cm under a fluorescent lamp (30 watts, Tru-lite fluorescent, Duro-test, Fairfield, NJ, with or without light-dark cycles) for a few days at 22 or 37 °C. Colonies were picked by forceps, blotted dry, and incubated with 18:2n-6. Nitrogen Powder of A. fumigatus and A. nidulans—A. nidulans was grown in liquid culture for 3 days at 37 °C (150 rpm), and A. fumigatus was grown for 24 h at 37 °C and then at room temperature (150 rpm) for 48 h. Mycelia (10–20 g) were harvested by filtration, washed with saline, and ground with liquid nitrogen to a fine powder, which was stored at –80 °C. The nitrogen powder was homogenized (glass-Teflon, 10 passes; 4 °C) in 10 volumes (w/v) of 0.1 mm KHPO4 buffer (pH 7.3), 2 mm EDTA, 0.04% Tween 20, centrifuged at 13, 000 × g (10 min, 4 °C), and used immediately for enzyme assay. Incubation with Mycelia—Mycelia (0.5–20 g) were incubated with 5 volumes (w/v) of 0.1 m NaBO3 buffer (pH 8.0 or 8.2) containing 18:2n-6 (0.5–1 mg/ml) for 5–6 h at 22 °C with shaking. The pH of the i