Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis
2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2022.102507
ISSN1083-351X
AutoresTakuya Teshima, Risa Funai, Takehito Nakazawa, Junya Ito, Toshihiko Utsumi, Pattana Kakumyan, Hiromi Mukai, Toyoshi Yoshiga, Ryutaro Murakami, Kiyotaka Nakagawa, Yoichi Honda, Kenji Matsui,
Tópico(s)Microbial metabolism and enzyme function
Resumo1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter–receiver ecological communication in mushrooms. 1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter–receiver ecological communication in mushrooms. 1-Octen-3-ol is a volatile compound with an earthy and mushroom-like organoleptic property that commonly occurs in nature, such as in mushrooms, molds, and moist air in a laurel forest. It is also found in human breath and sweat. It attracts biting insects, such as mosquitoes (1Potter C.J.H. Stop the biting: targeting a mosquito's sense of smell.Cell. 2014; 156: 878-881Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). An olfactory receptor specific to 1-octen-3-ol has been isolated from malaria-causing mosquitoes (2Takke W. Knols B.G.J. Odor-mediated behavior of afrotropical malaria mosquitoes.Annu. Rev. Entomol. 1999; 44: 131-157Crossref PubMed Scopus (497) Google Scholar). Despite its familiarity, the details of the biosynthesis of 1-octen-3-ol, as well as its ecological and physiological significance, are not fully understood. The biosynthetic pathway for 1-octen-3-ol formation in common mushrooms (Agaricus bisporus) has been elucidated by Wurzenberger and Grosch (3Wurzenberger M. Grosch W. The enzymic oxidative breakdown of linoleic acid in mushrooms (Psalliota bispora).Z. Lebensm. Unters. Forsch. 1982; 175: 168-190Crossref Scopus (88) Google Scholar, 4Wurzenberger M. Grosch W. The formation of 1-octen-3-ol from the 10-hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms (Psalliota bispora).Biochim. Biophys. Acta. 1984; 794: 25-30Crossref Scopus (113) Google Scholar, 5Wurzenberger M. Grosch W. Stereochemistry of the cleavage of the 10-hydroperoxide isomer of linoleic acid to 1-octen-3-ol by a hydroperoxide lyase from mushrooms (Psolliota bispora).Biochim. Biophys. Acta. 1984; 795: 163-165Crossref Scopus (72) Google Scholar). Linoleic acid is the substrate, and its stereospecific oxygenation yields the 10(S)-hydroperoxide of linoleic acid (10(S)HPODE) and subsequent cleavage yields (R)-(-)-1-octen-3-ol and 10-oxo-(E)-9-decenoic acid (Fig. 1). The involvement of the 10(S)-isomer as an intermediate to form 1-octen-3-ol from linoleic acid was also confirmed in Lentinula edodes (Shiitake mushroom) and Tricholoma matsutake (Matsutake mushroom) (6Akakabe Y. Matsui K. Kajiwara T. Stereochemical correlation between 10-hydroperoxyoctadecadienoic acid and 1-octen-3-ol in Lentinula edodes and Tricholoma Matsutake mushrooms.Biosci. Biotechnol. Biochem. 2005; 69: 1539-1544Crossref PubMed Scopus (21) Google Scholar, 7Garscha U. Oliw E.H. Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases.Anal. Biochem. 2007; 367: 238-246Crossref PubMed Scopus (40) Google Scholar). However, despite the efforts of many researchers, the enzymes involved in this biosynthetic pathway have not been identified for 40 years. Filamentous Ascomycota produce an array of oxylipin metabolites called precocious sexual inducer (psi) factors, which function as hormone-like signals. Psi-producing oxygenases (Ppos) are responsible for the biosynthesis of psi factors (8Tsitsigiannis D.I. Kowieski T.M. Zarnowski R. Keller N.P. Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans.Microbiology (Reading). 2005; 151: 1809-1821Crossref PubMed Scopus (140) Google Scholar, 9Tsitsigiannis D.I. Keller N.P. Oxylipins as developmental and host-fungal communication signals.Trends Microbiol. 2007; 15: 109-118Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 10Brodhun F. Feussner I. Oxylipins in fungi.FEBS J. 2011; 278: 1047-1063Crossref PubMed Scopus (141) Google Scholar, 11Oliw E.H. Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens.Fungal. Genet. Biol. 2021; 157: 103623Crossref PubMed Scopus (5) Google Scholar). Each Ppo is a fusion protein consisting of a cyclooxygenase (COX)-like domain at its N terminus and cytochrome P450-like domain at the C terminus. The COX domain catalyzes a dioxygenase (DOX) reaction with unsaturated fatty acids, such as linoleic acid and oleic acid, and the P450-like domain catalyzes a rearrangement reaction with the fatty acid hydroperoxide formed by the COX domain (10Brodhun F. Feussner I. Oxylipins in fungi.FEBS J. 2011; 278: 1047-1063Crossref PubMed Scopus (141) Google Scholar, 11Oliw E.H. Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens.Fungal. Genet. Biol. 2021; 157: 103623Crossref PubMed Scopus (5) Google Scholar). The filamentous Ascomycota have several subtypes of Ppos, such as PpoA, B, C, or D, which show diversity in the position of oxygenation on fatty acid substrates, catalyzed by the COX domain and the mode of the rearrangement of hydroperoxides thus formed, which is catalyzed by the P450 domain (10Brodhun F. Feussner I. Oxylipins in fungi.FEBS J. 2011; 278: 1047-1063Crossref PubMed Scopus (141) Google Scholar, 11Oliw E.H. Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens.Fungal. Genet. Biol. 2021; 157: 103623Crossref PubMed Scopus (5) Google Scholar). For example, PpoA, also called linoleate diol synthase, converts linoleic acid to its 8-hydroperoxide, which subsequently isomerizes to form 5,8-dihydroxy or 7,8-dihydroxy linoleic acid (12Brodowsky I.D. Hamberg M. Oliw E.H. A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannnomyces graminis.J. Biol. Chem. 1992; 267: 14738-14745Abstract Full Text PDF PubMed Google Scholar). PpoC from Aspergilli also consists of an N-terminal COX domain and C-terminal P450-like domain. The COX domain accounts for the formation of the 10-hydroperoxide of linoleic acid but the P450-like domain does not rearrange hydroperoxide; thus, PpoC almost exclusively forms the 10-hydroperoxide of linoleic acid (13Brodhun F. Schneider S. Göbel C. Hornung E. Feussner I. PpoC from Aspergillus nidulans is a fusion protein with only one active haem.Biochem. J. 2010; 425: 553-565Crossref PubMed Scopus (45) Google Scholar). Because of the same position of the hydroperoxide group in linoleic acid, PpoC-like enzymes are expected to be involved in the production of 1-octen-3-ol in filamentous Ascomycota. Disruption of ppoC-like genes in Podospora anserina and Aspergillus luchuensis diminishes their ability to produce 1-octen-3-ol (14Ferrari R. Lacaze I. Le Faouder P. Bertrand-Michel J. Oger C. Galano J.M. et al.Cyclooxygenase and lipoxygenase are used by the fungus Podospora anserina to repel nematodes.Biochim. Biophys. Acta. 2018; 1862: 2174-2182Crossref Scopus (7) Google Scholar, 15Kataoka R. Watanabe T. Yano S. Mizutani O. Yamada O. Kasumi T. et al.Aspergillus luchuensis fatty acid oxygenase ppoC in necessary for 1-octen-3-ol biosynthesis in rice koji.J. Biosci. Bioeng. 2020; 129: 192-198Crossref PubMed Scopus (10) Google Scholar). However, it has not been confirmed whether the ppoC-like genes of P. anserina and A. luchuensis are directly involved in the formation of 1-octen-3-ol because the catalytic properties of these gene products have not been studied. Furthermore, the configuration of the PpoC reaction products from filamentous ascomycetes analyzed to date was 10(R)-hydroperoxide (7Garscha U. Oliw E.H. Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases.Anal. Biochem. 2007; 367: 238-246Crossref PubMed Scopus (40) Google Scholar), which was not cleaved to form 1-octen-3-ol by the crude extract prepared from common mushrooms (5Wurzenberger M. Grosch W. Stereochemistry of the cleavage of the 10-hydroperoxide isomer of linoleic acid to 1-octen-3-ol by a hydroperoxide lyase from mushrooms (Psolliota bispora).Biochim. Biophys. Acta. 1984; 795: 163-165Crossref Scopus (72) Google Scholar). To date, four genes with substantial similarities with ascomycete ppos have been reported in Basidiomycota, such as Ustilago maydis and Rhizoctonia solani (16Huber S.M.F.E. Lottspeich F. Kämper J. A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis.Mol. Genet. Genomics. 2002; 267: 757-771Crossref PubMed Scopus (38) Google Scholar, 17Oliw E.H. Biosynthesis of oxylipins by Rhizoctonia solani with allene oxide and oleate 8S,9S-diol synthase activities.Lipids. 2018; 53: 527-537Crossref PubMed Scopus (6) Google Scholar); however, the properties and functions of the enzymes encoded by these basidiomycete genes have never been studied. In this study, we identified two genes, Ccdox1 and Ccdox2, in the genome of the model basidiomycete Coprinopsis cinerea, which shares homology with ppos from filamentous ascomycetes. C. cinerea, commonly known as the gray shag, is a model multicellular basidiomycete that completes its entire life cycle through a sexual cycle within 2 weeks in the laboratory (18Kües U. Life history and developmental processes in the basidiomycete Coprinus cinereus.Microbiol. Mol. Biol. Rev. 2000; 64: 316-353Crossref PubMed Scopus (360) Google Scholar). A rich genetic resource, with genome sequences, morphological and developmental mutants, and DNA markers, is available for the mushroom. C. cinerea is relatively easy to genetically transform, and a procedure for targeted gene disruption has been established (19Stajich J.E. Wilke S.K. Ahrén D. Pukkila P.J. Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 11889-11894Crossref PubMed Scopus (315) Google Scholar). We characterized the enzymatic properties of recombinant CcDOX1 and CcDOX2 expressed in insect cells. Ccdox1 gene was disrupted through homologous recombination to confirm its involvement in 1-octen-3-ol formation. The ecophysiological role of 1-octen-3-ol may confer the benefit of sporophyte dispersal by attracting mosquitoes and flies. 1-Octen-3-ol has also been reported to have behavioral suppression and repellent effects on several arthropods and nematodes (20Inamdar A.A. Morath S. Bennett J.W. Fungal volatile organic compounds: more than just a funky smell?.Annu. Rev. Microbiol. 2020; 74: 101-116Crossref PubMed Scopus (25) Google Scholar), indicating its potential involvement in the defense against fungivores. However, there is no clear evidence confirming the contribution of 1-octen-3-ol to Basidiomycota. In this study, a C. cinerea strain deficient in the ability to form 1-octen-3-ol was used to examine the ecological and/or physiological significance of 1-octen-3-ol. 1-Octen-3-ol was barely detected in the intact mycelia of C. cinerea (strain ku3-24) grown on yeast extract-malt extract-glucose (YMG) agar plates, but the formation of considerable amounts of 1-octen-3-ol along with 3-octanone, 1-octanol, and 3-methylbutanal was observed within 30 min of damaging the mycelia with freeze-thaw treatment (Fig. 2A). The rapid formation of 1-octen-3-ol was effectively suppressed in the absence of molecular oxygen (Fig. 2B), indicating de novo biosynthesis of 1-octen-3-ol from linoleic acid. Rapid formation of 1-octen-3-ol was also observed when the mycelia were disrupted with a bead crusher, and the formation was suppressed by the addition of Ca2+ chelating reagents, such as BAPTA or EGTA (Fig. 2C). Acetylsalicylic acid, a typical cyclooxygenase inhibitor, also suppressed 1-octen-3-ol formation; however, only minimal suppression was observed with the cyclooxygenase inhibitors ibuprofen and mefenamic acid (Fig. 2D). Because Ppo and lipoxygenase (LOX) have been reported in fungi as enzymes catalyzing the dioxygenation of fatty acids to yield the hydroperoxide derivative (10Brodhun F. Feussner I. Oxylipins in fungi.FEBS J. 2011; 278: 1047-1063Crossref PubMed Scopus (141) Google Scholar), we assumed that an enzyme similar to Ppo or LOX participated in the first fatty acid oxygenation step in the biosynthetic pathway to form 1-octen-3-ol. When manganese LOX in the filamentous ascomycete Gaeumannomyces graminis var. avenae (oat-take-all root rot fungus) (GenBank, AAK81882.2) (21Hörnsten L. Su C. Osbourn A.E. Hellman U. Oliw E.H. Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family.Eur. J. Biochem. 2002; 269: 2690-2697Crossref PubMed Scopus (39) Google Scholar) was used as a query for BLASTP search with the C. cinerea genome database (Coprinopsis cinerea AmutBmut pab1-1 v1.0 in MycoCosm) (22Grigoriev I.V. Nikitin R. Haridas S. Kuo A. Ohm R. Otillar R. et al.MycoCosm portal: gearing up for 1000 fungal genomes.Nucleic Acids Res. 2014; 42: D699-D704Crossref PubMed Scopus (768) Google Scholar), no protein with a substantial similarity was detected. When the same BLASTP search was performed with Aspergillus nidulans PpoC protein (AAT36614) as the query, two proteins with protein IDs 423716 and 398037 (GenBank: EAU90460.2 and EAU86789.2, respectively) were found, with E-values smaller than 1.0e−100. They were tentatively named CcDOX1 and CcDOX2. CcDOX1 and CcDOX2 are 1066 and 1118 amino acids long, respectively, and share 38% identity and 54% homology. TargetP-2.0 analyses (http://www.cbs.dtu.dk/services/TargetP/) showed that both proteins contained neither a signal peptide nor a mitochondrial transit peptide. InterProScan (https://www.ebi.ac.uk/interpro/search/sequence/) with CcDOX1 indicated that it consists of an N-terminal domain belonging to the animal heme peroxidase family (IPR037120) and a C-terminal domain that was classified as a sequence homologous to the cytochrome P450 superfamily (IPR036396), although the sequence was not classified as a member of any protein family specified in InterPro (Fig. 3A). This was mostly the case with CcDOX2, but a short segment of its C-terminal domain was classified as a member of the cytochrome P450 family (IPR001128) (Fig. 3A). The catalytic Tyr (Tyr-395 and Tyr-407 in CcDOX1 and CcDOX2, respectively) and distal and proximal His (His-217/His-398 and His-227/His-410 in CcDOX1 and CcDOX2, respectively) essential for binding heme are highly conserved as found with animal COXs (23Marnett L.J. Rowlinson S.W. Goodwin D.C. Kalgutkar A.S. Lanzo C.A. Arachidonic acid oxygenation by COX-1 and COX-2.J. Biol. Chem. 1999; 274: 22903-22906Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar) (Fig. S1). They are conserved at the apparently suitable positions in the protein sequences in the N-terminal domains of CcDOX1/2. The Arg residue critical for binding fatty acid substrates (Arg-106 in mouse COX2) was not found, and Ser, which is the target of nonsteroidal anti-inflammatory drugs (Ser-516 in mouse COX2) (23Marnett L.J. Rowlinson S.W. Goodwin D.C. Kalgutkar A.S. Lanzo C.A. Arachidonic acid oxygenation by COX-1 and COX-2.J. Biol. Chem. 1999; 274: 22903-22906Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar), was replaced with Thr in CcDOX1/2 (Fig. S1). The signature of the C-terminal domain of P450 is not distinct. The ExxR motif (24Brodhun F. Göbel C. Hornung E. Feussner I. Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450.J. Biol. Chem. 2009; 284: 11792-11805Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), which is one of the most conserved residues in the K-helix of Cyt P450 enzymes, is conserved in CcDOX1/2; however, the heme signature motif (FxxGx(H/R)xCxG motif) (24Brodhun F. Göbel C. Hornung E. Feussner I. Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450.J. Biol. Chem. 2009; 284: 11792-11805Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) is quite different in CcDOX1/2. For example, the His residue, which has been shown to be essential for hydroperoxide isomerase activity in A. nidulans PpoA (His1004) (24Brodhun F. Göbel C. Hornung E. Feussner I. Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450.J. Biol. Chem. 2009; 284: 11792-11805Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and the Cys residue, crucial for P450 activity as the fifth heme iron ligand through the heme-thiolate bond (25Denisov I.G. Makris T.M. Sligar S.G. Schlichting I. Structure and chemistry of cytochrome P450.Chem. Rev. 2005; 105: 2253-2277Crossref PubMed Scopus (1567) Google Scholar), are replaced by Phe/Thr and Tyr/Leu in CcDOX1/2, respectively (Fig. S2). Substitution of these amino acid residues, which are essential for P450 catalysis, is also evident in A. nidulans PpoC, which has no ability to rearrange the fatty acid hydroperoxide depending on P450 catalysis (13Brodhun F. Schneider S. Göbel C. Hornung E. Feussner I. PpoC from Aspergillus nidulans is a fusion protein with only one active haem.Biochem. J. 2010; 425: 553-565Crossref PubMed Scopus (45) Google Scholar). Hereafter, the C-terminal domain of CcDOX1/2 is referred to as a P450-related domain. The CcDOX1 sequence was used as a query for BLASTP analyses in MycoCosm (22Grigoriev I.V. Nikitin R. Haridas S. Kuo A. Ohm R. Otillar R. et al.MycoCosm portal: gearing up for 1000 fungal genomes.Nucleic Acids Res. 2014; 42: D699-D704Crossref PubMed Scopus (768) Google Scholar) against the protein databases of the genome sequences of representative species belonging to diverse classes in Basidiomycota, namely, A. bisporus (Agaricales), Pleurotus ostreatus (Agaricales), Schizophyllum commune (Agaricales), R. solani (Cantharellales) (17Oliw E.H. Biosynthesis of oxylipins by Rhizoctonia solani with allene oxide and oleate 8S,9S-diol synthase activities.Lipids. 2018; 53: 527-537Crossref PubMed Scopus (6) Google Scholar), Serpula lacrymans (Boletales), Fomitopsis pinicola (Polyporales), Rickenella mellea (Rickenella), and U. maydis (Ustilaginomycotina) (16Huber S.M.F.E. Lottspeich F. Kämper J. A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis.Mol. Genet. Genomics. 2002; 267: 757-771Crossref PubMed Scopus (38) Google Scholar). Proteins with a significant similarity (<1.0e−5) were chosen and a phylogenetic tree was constructed along with the protein sequences of representative DOX-P450 fusion proteins (Ppos) from Ascomycota (26Oliw E.H. Argó M. Chen Y. Jernerén F. A new class of fatty acid allene oxide formed by the DOX-P450 fusion proteins of human and plant pathogenic fungi, C. immitis and Z. tririci.J. Lipid Res. 2016; 57: 1518-1528Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) (Fig. 3B). The proteins were divided into four major clades (clade I to IV), three of which (clade I, II, and III) consisted of proteins in Ascomycota, and the other one (clade IV) consisted of those in Basidiomycota. The proteins found in Basidiomycota were further divided into three major clades (subclades a, b, and c), where CcDOX1 and CcDOX2 were located in subclades a and b, respectively. The proteins in subclades a and b had the N-terminal DOX domain and the C-terminal P450-related domain. The heme signature motifs (FxxGx(H/R)xCxG) of almost all of the proteins in subclades a and b were not conserved (Fig. S3). The C-terminal domains of those in subclade c consisted of proteins that harbored an apparently complete heme signature motif. Ssp1 protein is highly expressed in the teliospores of U. maydis (Ustilaginomycotina) (AAL38020.1) (16Huber S.M.F.E. Lottspeich F. Kämper J. A gene that encodes a product with similarity to dioxygenases is highly expressed in teliospores of Ustilago maydis.Mol. Genet. Genomics. 2002; 267: 757-771Crossref PubMed Scopus (38) Google Scholar) and did not belong to any subclade. It has been reported that recombinant A. nidulans PpoC (AnPpoC) is unstable when the enzyme is expressed in Escherichia coli (13Brodhun F. Schneider S. Göbel C. Hornung E. Feussner I. PpoC from Aspergillus nidulans is a fusion protein with only one active haem.Biochem. J. 2010; 425: 553-565Crossref PubMed Scopus (45) Google Scholar). It has also been reported that recombinant G. graminis linoleate diol synthase (GgLDS) expressed with Pichia pastoris showed a product specificity different from that observed for the enzyme prepared from the mycelia of G. graminis (27Garscha U. Oliw E. Pichia expression and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase.Biochem. Biophys. Res. Commun. 2008; 373: 579-583Crossref PubMed Scopus (9) Google Scholar), while recombinant GgLDS expressed with insect cells (Sf9) showed the same product specificity (28Garscha U. Oliw E. Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases.FEBS Lett. 2008; 582: 3547-3551Crossref PubMed Scopus (18) Google Scholar). Accordingly, in this study, we chose insect cells as the host for expression of recombinant CcDOXs. Recombinant proteins encoded by Ccdox1 and 2 were transiently expressed in BmN4 cells derived from the silkworm Bombyx mori with or without enhanced GFP (EGFP) fused at their C-terminals. To evaluate the enzymatic activity of the recombinant proteins, the cell lysate expressing the respective proteins was reacted with linoleic acid, and the products were analyzed in the negative-enhanced mass spectrum mode of LC-MS/MS. Chromatograms were drawn by extracting the molecular ions or fragment ions associated with the oxygenated products of linoleic acid, such as hydroperoxides, hydroxides, diols, or epoxyalcohols, which were expected based on the reaction of the Ascomycota Ppo enzymes (10Brodhun F. Feussner I. Oxylipins in fungi.FEBS J. 2011; 278: 1047-1063Crossref PubMed Scopus (141) Google Scholar). Accordingly, the formation of the hydroperoxide derivative of linoleic acid (m/z 293, [C18H32O4-H3O+]−) and the hydroxide of linoleic acid (m/z 295, [C18H32O3-H+]−) was evident in the crude lysate prepared from the insect cells expressing all four recombinant CcDOXs (CcDOX1 or CcDOX2, with/without EGFP) (Fig. S4). No sign suggestive of compounds other than hydroperoxide and hydroxide of linoleic acid was detected. Peaks 3 and 6 apparent with m/z 295 were tentatively assigned as 10-hydroperoxide and 8-hydroperoxide of oleic acid ([C18H34O4-H3O+]−), respectively (Fig. S5). They were not detected when purified CcDOX1/2 was reacted with linoleic acid. They were most likely formed from oleic acid endogenous to insect cells. Interestingly, the hydroperoxides of oleic acid were not found in the insect cells before disruption. This suggests that either the recombinant CcDOXs in the insect cells were in latent states and were activated upon cell disruption or that the hydrolysis of membrane lipids facilitated by cell disruption supplied free oleic acid as a substrate for the recombinant CcDOXs. Based on the chromatograms, fusion of the EGFP sequence to the C terminus of either CcDOX1 or CcDOX2 had little effect on the activity and product specificity of the reaction. The hydroxides were likely formed by unknown components derived from the cell lysates because the reduction was not observed when purified CcDOX1/2 was used for product analysis. Recombinant CcDOX1 and CcDOX2 proteins were purified by immunoprecipitation with an anti-GFP antibody (Fig. S6). Linoleic acid was added to the purified recombinant CcDOX1 and CcDOX2, and the products were extracted, reduced with triphenylphosphine, and analyzed by LC-MS/MS in the negative enhanced product ion mode (Fig. S7). With this analysis, a peak tentatively assigned as 10-hydroxides or 8-hydroxides of linoleic acid based on the fragment ion diagnostic to the position of the hydroxide group (7Garscha U. Oliw E.H. Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases.Anal. Biochem. 2007; 367: 238-246Crossref PubMed Scopus (40) Google Scholar) was detected as the product formed by CcDOX1 or CcDOX2, respectively. To examine the expression of Ccdox genes, C. cinerea was grown in liquid YMG medium in a static culture. The mycelia grew rapidly under the growth conditions used, and the growth reached a plateau at 4 days after inoculation (Fig. 4A). The amount of 1-octen-3-ol produced by freeze-thaw treatment gradually increased from day 4, it reached a peak on day 12, and then decreased thereafter (Fig. 4B). The transcript level of Ccdox1 mostly followed the amount of 1-octen-3-ol but remained high even on day 16 (Fig. 4C). The developmental time course of Ccdox2 expression was distinct from those of Ccdox1 and of the 1-octen-3-ol formation ability. The expression level of Ccdox2 reached a peak at 8 days after transplanting. Thereafter, levels decreased to almost undetectable levels on day 16. As the expression profile of Ccdox1 gene and the result that the main product of the recombinant CcDOX1 from linoleic acid was tentatively assigned as 10HPODE, which was reported to be the intermediate in the biosynthetic pathway to form 1-octen-3-ol in basidiomycetes, CcDOX1 was chosen for further extensive analysis. We used tandem MS analysis in the presence of sodium ions, and each isomer of the fatty acid hydroperoxide was identified and quantified using the corresponding authentic specimen (29Ito J. Mizuochi S. Nakagawa K. Kato S. Miyazawa T. Tandem mass spectrometry analysis of linoleic acid and arachidonic acid hydroperoxides via promotion of alkali metal adduct formation.Anal. Chem. 2015; 87: 4980-4987Crossref PubMed Scopus (36) Google Scholar, 30Ito J. Komuro M. Parida I.S. Shimizu N. Kato S. Meguro Y. et al.Evaluation of lipid oxidation mechanisms in beverages and cosmetics via analysis of lipid hydroperoxide isomers.Sci. Rep. 2019; 9: 7387Crossref PubMed Scopus (17) Google Scholar). The main product formed from linoleic acid by immunopurified recombinant CcDOX1-EGFP was confirmed to be 10HPODE (Fig. 5A). Other isomers, such as 9, 12, and 13HPODE, were barely detected. The positional specificity in terms of the oxygen insertion was strict, and 10-hydroperoxides were the major product, even with oleic acid or α-linolenic acid. Using chiral phase chromatography (31Ito J. Shimizu N. Kobayashi E. Hanzawa Y. Otoki Y. Kato S. et al.A novel chiral stationary phase LC-MS/MS method to evaluate oxidation mechanisms of edible oils.Sci. Rep. 2017; 7: 10026Crossref PubMed Scopus (25) Google Scholar), the two enantiomers of 10HPODE prepared by photo-oxidation of linoleic acid were separated (Fig. 5B). The product formed by recombinant CcDOX1 from linoleic acid showed only one peak with the same retention time as that of 10(S)HPODE prepared with recombinant Nostoc punctiforme dioxygenase (NpDOX) (32Brash A.R. Niraula N.P. Boeglin W.E. Mashhadi Z. An ancient relative of cyclooxygenase in cyan
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