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A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans

2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês

10.1074/jbc.m705068200

1083-351X

Mareike D. Hoffmann, Ellen Hornung, Silke Busch, Nina Kassner, Philipp Ternes, Gerhard H. Braus, Ivo Feußner,

Enzyme Catalysis and Immobilization

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Δ12 desaturase and a processive bifunctional oleoyl-Δ12/linoleoyl-ω3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Δ12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Δ12/ω3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model. Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Δ12 desaturase and a processive bifunctional oleoyl-Δ12/linoleoyl-ω3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Δ12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Δ12/ω3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model. Metabolic processes are often controlled by the presence of highly specific enzymes. An interesting model to study enzyme specificity is a fatty acid desaturase, which introduces double bonds at specific positions into acyl chains (1Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar). Desaturase enzymes differ in substrate- and regioselectivity (2Heinz E. Lipid Metabolism in Plants.in: Moore Jr., T.S. CRC Press, London1993: 33-89Google Scholar). The strict substrate- and regiospecificities are of physiological relevance, because the major mechanism controlling the biophysical properties of a membrane, aside from changing its overall lipid class composition, is the modification of acyl chain length, position, and number of the double bonds in glycerolipids (3Somerville C. Browse J. Jaworski J.G. Ohlrogge J. Biochemistry & Molecular Biology of Plants.in: Buchanan B.B. Gruissem W. Jones R.L. American Society of Plant Physiologists, Rockville, MD. 2000: 456-527Google Scholar). Fatty acid desaturases may also control the acyl composition of seed storage lipids, which is of considerable economic interest. Despite the importance of desaturases, knowledge about the determinants of their enzymatic specificity is still in its infancy. The solution of the three-dimensional crystal structure of the soluble castor bean and ivy stearoyl-ACP 2The abbreviations used are:ACPacyl carrier proteinFAMEsfatty methyl estersGMMglucose minimal mediumORFopen reading framePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineERendoplasmic reticulumDMOX4,4-dimethyloxazoline 2The abbreviations used are:ACPacyl carrier proteinFAMEsfatty methyl estersGMMglucose minimal mediumORFopen reading framePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineERendoplasmic reticulumDMOX4,4-dimethyloxazoline-desaturases has brought some insight into the structure/function relationship involved in the determination of specificity of soluble desaturase enzymes (4Lindqvist Y. Huang W.J. Schneider G. Shanklin J. EMBO J. 1996; 15: 4081-4092Crossref PubMed Scopus (385) Google Scholar, 5Cahoon E.B. Lindqvist Y. Schneider G. Shanklin J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4872-4877Crossref PubMed Scopus (142) Google Scholar, 6Guy J.E. Whittle E. Kumaran D. Lindqvist Y. Shanklin J. J. Biol. Chem. 2007; 282: 19863-19871Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, the majority of desaturase enzymes reside within membranes, and up to now the principles of membrane-bound desaturase specificity are not understood. Moreover, structural information on membrane-bound desaturases is limited, so only hydropathy and topology analyses are available, predicting the formation of four transmembrane domains and either one or two membrane-peripheral protein domains (7Diaz A.R. Mansilla M.C. Vila A.J. de Mendoza D. J. Biol. Chem. 2002; 277: 48099-48106Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Stukey J.E. McDonough V.M. Martin C.E. J. Biol. Chem. 1990; 265: 20144-20149Abstract Full Text PDF PubMed Google Scholar, 9Man W.C. Miyazaki M. Chu K. Ntambi J.M. J. Biol. Chem. 2006; 281: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Three highly conserved histidine-rich motifs are essential for catalysis (1Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar, 10Sperling P. Ternes P. Zank T.K. Heinz E. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 73-95Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), and it was proposed that these histidine clusters coordinate two iron atoms in the active site (11Fox B.G. Lyle K.S. Rogge C.E. Acc. Chem. Res. 2004; 37: 421-429Crossref PubMed Scopus (141) Google Scholar). acyl carrier protein fatty methyl esters glucose minimal medium open reading frame phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine endoplasmic reticulum 4,4-dimethyloxazoline acyl carrier protein fatty methyl esters glucose minimal medium open reading frame phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine endoplasmic reticulum 4,4-dimethyloxazoline The clearly related genes for membrane-bound desaturases have developed a broad range of catalytic diversity of the encoded desaturase enzymes during evolution (12Shanklin J. Curr. Opin. Plant Biol. 2000; 3: 243-248Crossref PubMed Scopus (18) Google Scholar). Depending on the enzyme studied, introduction of a double bond can occur counting carbons from the carboxyl end or from the methyl terminus of the acyl chain. Other enzymes may use a pre-existing double bond as a reference point for subsequent desaturation (2Heinz E. Lipid Metabolism in Plants.in: Moore Jr., T.S. CRC Press, London1993: 33-89Google Scholar). Also the carbon chain length (2Heinz E. Lipid Metabolism in Plants.in: Moore Jr., T.S. CRC Press, London1993: 33-89Google Scholar) or the specific lipid head group of the glycerolipid to which the substrate fatty acid is esterified can be distinguished (13Heilmann I. Pidkowich M.S. Girke T. Shanklin J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10266-10271Crossref PubMed Scopus (72) Google Scholar). One conceivable model how changes in regiospecificity may have developed in evolution is by gene duplication and random accumulation of mutations in the catalytic portion of the enzymes to produce first a broader and then a different specificity (10Sperling P. Ternes P. Zank T.K. Heinz E. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 73-95Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 12Shanklin J. Curr. Opin. Plant Biol. 2000; 3: 243-248Crossref PubMed Scopus (18) Google Scholar). Changes to an enzyme regiospecificity typically require between two and six specific alterations at key locations along the amino acid chain (6Guy J.E. Whittle E. Kumaran D. Lindqvist Y. Shanklin J. J. Biol. Chem. 2007; 282: 19863-19871Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Broadwater J.A. Whittle E. Shanklin J. J. Biol. Chem. 2002; 277: 15613-15620Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). According to the described model the first step toward a new specificity of an enzyme is the accumulation of mutations in the corresponding gene, which result in decreased enzyme specificity and lead to broader substrate acceptance and/or formation of multiple products (12Shanklin J. Curr. Opin. Plant Biol. 2000; 3: 243-248Crossref PubMed Scopus (18) Google Scholar). The simplest case of multi-functionality is represented by bifunctional enzymes. Examples include the oleate 12-hydroxylase/desaturase from the flowering plant Lesquerella fendleri (15Broun P. Boddupalli S. Somerville C. Plant J. 1998; 13: 201-210Crossref PubMed Scopus (139) Google Scholar), a Δ6 acetylenase/desaturase from the moss Ceratodon purpureus (16Sperling P. Lee M. Girke T. Zähringer U. Stymne S. Heinz E. Eur. J. Biochem. 2000; 267: 3801-3811Crossref PubMed Scopus (72) Google Scholar), a Δ5/6 desaturase from the fish Danio rerio (17Hastings N. Agaba M. Tocher D.R. Leaver M.J. Dick J.R. Sargent J.R. Teale A.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14304-14309Crossref PubMed Scopus (276) Google Scholar), an acyl-ACP desaturase from the flowering plant Hedera helix, which can synthesize 16- and 18-carbon monoene and diene products (18Whittle E. Cahoon E.B. Subrahmanyam S. Shanklin J. J. Biol. Chem. 2005; 280: 28169-28176Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and Δ12/ω3 desaturases from the fungi Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea (19Damude H.G. Zhang H. Farrall L. Ripp K.G. Tomb J-F. Hollerbach D. Yadav N.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9446-9451Crossref PubMed Scopus (136) Google Scholar). The determinants of altered desaturase specificity may be identified by the comparison of a strictly specific monofunctional to a bifunctional enzyme. Here we compare the specific An2 oleoyl-Δ12 desaturase (19Damude H.G. Zhang H. Farrall L. Ripp K.G. Tomb J-F. Hollerbach D. Yadav N.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9446-9451Crossref PubMed Scopus (136) Google Scholar) to the bifunctional An1 oleoyl-Δ12/linoleoyl-ω3 desaturase from Aspergillus nidulans. Heterologous expression of the Δ12/ω3 desaturase gene in yeast and in Arabidopsis seeds demonstrated its broad ω6 substrate specificity and especially its bifunctionality, similar to that of reported bifunctional enzymes (19Damude H.G. Zhang H. Farrall L. Ripp K.G. Tomb J-F. Hollerbach D. Yadav N.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9446-9451Crossref PubMed Scopus (136) Google Scholar). By using a domain-swapping approach between the genes for the two enzymes, domains responsible for the broadened substrate specificity and altered regioselectivity were identified and reside in a small membrane-peripheral region in close proximity to the active site. Materials—Restriction enzymes and DNA-modifying enzymes were obtained from MBI Fermentas. Standards of fatty acids as well as all other chemicals were from Sigma; methanol, hexane, 2-propyl alcohol (all HPLC grade) were from Baker. Fatty acids were either obtained from Cayman Chemicals or Larodan. Basic molecular biological and biochemical techniques were performed as described (20Ausubel F.M. Brent R.E. Kingston D.D. Seidmann J.R. Smith J.A. Struhl K. Current Protocols in Molecular Biology.in: Green Publishing Associates and John Wiley and Sons Inc., New York1993Google Scholar). Cultivation of A. nidulans—A. nidulans (strain FGSC A4) was cultivated in glucose minimal medium (GMM) (21Calvo A.M. Gardner H.W. Keller N.P. J. Biol. Chem. 2001; 276: 25766-25774Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) at 37 °C. For vegetative growth cultures were cultivated in liquid GMM at 37 °C in the light and shaken vigorously at 220 rpm. For sexual growth cultures were incubated in liquid GMM at 37 °C in the dark. Mycelium was harvested by vacuum filtration through sterile filter paper. Isolation and Cloning of Desaturase Genes—Total RNAs were extracted from mycelia obtained from vegetative growth or sexual development using TRIzol reagent (Invitrogen). First strand cDNA was synthesized from total RNA using M-MuLV Reverse Transcriptase (MBI Fermentas). The desaturase genes An2 (GenBank™ accession no. XP_658641) and An1 (GenBank™ accession no. XP_664808) were isolated by PCR amplification using the Expand High Fidelity polymerase (Roche Diagnostics) and a standard PCR protocol (2 min at 94 °C, 10 cycles of 30 s at 94 °C, 30 s at 55 °C, 60 s at 72 °C, followed by 15 cycles of 30 s at 94 °C, 30 s at 55 °C, and 60 s at 72 °C; and a terminal extension step of 5 min at 72 °C) with primers 5′-GAA TTCATGGCTGCAACTGCAACAACCC-3′ (An2.forward)/5′-GGCGGCCGCCTATTCCGCTTTGGCACCCTTC-3′ (An2. reverse), and (An1.forward) 5′-GAA TTC ATG GCC TCG GAT GCG GGC AAG-3′/(An1.reverse) 5′-GGCGGCCGCTTAGTTAGGCTTGGTCAGCTTAATG-3′, respectively, introducing EcoRI sites upstream of the start codons and NotI sites downstream of the stop codons. The ORFs were cloned into pGEM-T (Promega) and moved as EcoRI/NotI fragments into the yeast expression vector pESC-HIS (Stratagene), yielding pESC-An2 and pESC-An1. Preparation of Δ12- and Δ12/ω3 Desaturase Constructs—The ORFs of An2 and An1 desaturases were modified using the Expand High Fidelity polymerase (Roche Diagnostics) and a standard PCR protocol (see above) with primers: 5′-GGGAATTCGCTATGGCCTCGGATGCGGGCAAG-3′(An2for)/5′-GTCACTCGAGGTTAGGCTTGGTCAGCTT-3′ (An2rev) and (An1for) 5′-GGGAATTCGCTATGGCTGCAACTGCAACAACC-3′/(An1rev) 5′-GTCACTCGAGTTCCGCTTTGGCACCCTTCTT-3′, respectively, introducing EcoRI sites upstream of the start codons (in boldface) and removing the stop codons, respectively, introducing XhoI sites downstream of the terminal triplets. The ORFs were cloned into the amplification plasmid pGEM-T (Promega) and moved as EcoRI/XhoI fragments into the yeast expression vector pYES2/CT (Invitrogen) in-frame with the sequence encoding a C-terminal V5-tag, yielding pYES2/CT-An2 and pYES2/CT-An1. Cloning and Vector Construction for Production of Transgenic Arabidopsis Plants—The cDNA ORF of AnΔ12/ω3 was moved as an EcoRI/NotI-fragment into the Gateway Entry vector pUC18-Entry (22Hornung E. Krueger C. Pernstich C. Gipmans M. Porzel A. Feussner I. Biochim. Biophys. Acta. 2005; 1738: 105-114Crossref PubMed Scopus (62) Google Scholar). The insert was introduced by Gateway cloning (Invitrogen) into the binary plasmid pCAMBIA modified for resistance to ammonium glufosinate and seed-specific expression using the USP promoter (22Hornung E. Krueger C. Pernstich C. Gipmans M. Porzel A. Feussner I. Biochim. Biophys. Acta. 2005; 1738: 105-114Crossref PubMed Scopus (62) Google Scholar). The construct was transformed into chemically competent Agrobacteria (strain EH105), and introduced into Arabidopsis thaliana ecotype Columbia by floral dipping (23Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). T3-seeds were collected from individual T2 plants resistant to ammonium glufosinate and analyzed individually by GC. Engineering Hybrid Genes—According to results from hydropathy analysis, the desaturases An2 and An1 were divided into nine domains, A1–I1, and A2–I2, respectively (see Fig. 2, A and B), that were systematically exchanged between the two desaturase proteins to delineate determinants of enzymatic specificity. Domain swap constructs between An2 and An1 were generated by overlap extension PCR (24Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2614) Google Scholar). In a first step, domains A1–I1 were amplified individually from an An1 cDNA template with overlapping ends using primers specified in Table 1. Similarly, domains A2–I2 were amplified from An2 cDNA (see Table 1). cDNAs encoding the respective truncated acceptor desaturase domains were amplified from An2 and An1 cDNA using primers also listed in Table 1. Amplifications were carried out with Expand High Fidelity polymerase (Roche Diagnostics) and a protocol of 2 min at 94 °C, 10 cycles of 15 sat 94 °C, 30 s at 68 °C, 70 s at 72 °C, followed by 20 cycles of 15 s at 94 °C, 30 s at 70 °C, and 70 s at 72 °C; and a terminal extension step of 7 min at 72 °C. Amplicons were fused in a subsequent PCR reaction, using primers An2for/An2rev and An1for/An1rev, respectively, and the protocol specified for the first step, generating the hybrid gene constructs of the An2- and An1 desaturases indicated in Table 1. The hybrid genes were cloned into the amplification plasmid pGEM-T (Promega) and moved as EcoRI/XhoI fragments into the yeast expression vector pYES2/CT (Invitrogen) in-frame with the sequence encoding a C-terminal V5 tag.TABLE 1Oligonucleotide primers used to construct hybrid genes of A. nidulans bifunctional Δ12/ω3-desaturases (An1) and monofunctional Δ12 (An2) fatty acid desaturasesPrimerSequenceBifunctional Δ12/ω3-Desaturase, An1An1for5′-GGGAATTCGCTATGGCTGCAACTGCAACAACC-3′An21rev5′-GCGAGATCACGGAAGACGTAGGCAGAGGAAA-3′An1B1for5′-TCAGGAGTCTGTCCTATGTCGTCCGCGATCTCC-3′An1B1rev5′-TGAGCAAGAACCCAAATGATTCCGGTGCC-3′An1C1for5′-GGTCTGTTCGGTACCGGTATCTGGATCCTC-3′An1C1rev5′-GATCCAGCCAACAGTGTCGTTCCACGTTTG-3′An1D1for5′-ACTTCCAAGGTGCTCAACGATGTCGTCGGA-3′An1D1rev5′-CTTGCCGTGAGAGATCTTCCAGCTGAAA-3′An1E1for5′-GTCCCCTATTTCTCGTGGAAGATCACGCA-3′An1E1rev5′-CAGGATGGAGTGGGTGGCGACGGGCGTGTCCTC-3′An1F1for5′-GAGACCCCCTTGGCCACTTACAACCTTATTAGCCTA-3′An1F1rev5′-TGTCGTGACCGGTAACGTTACAGAGATACATCTG-3′An1G1for5′-CCCTTGTACCTGCTCACCTTTTACGTTAGCGCC-3′An1G1rev5′-GGCGGTGATGGCGAGGCCCGAAATCGCAATCAG-3′An1H1for5′-TCATCATTCTGAGTGATATCGACCTGGGGTTGCTG-3′An1H1rev5′-GCTGGTAGTGGGGGGAGAGTGGGGTGTGTGTGG-3′An1I1for5′-GCCATCACCTACCTCCAGCACACACACCCCT-3′An1rev5′-GTCACTCGAGTTCCGCTTTGGCACCCTTCTT-3′Monofunctional Δ12-Desaturase, An2An2for5′-GGGAATTCGCTATGGCCTCGGATGCGGGCAAG-3′An2A2rev5′-GGAGATCGCGGACGACATAGGACAGACTCCTGA-3′An2B2for5′-TTTCCTCTGCCTACGTCTTCCGTGATCTCGC-3′An2B2rev5′-GAGGATCCAGATACCGGTACCGAACAGCCA-3′An2C2for5′-GGCACCGGAATCATTTGGGTTCTTGCTCA-3′An2C2rev5′-TCCGACCACATCGTTGAGCACCTTGGAAGT-3′An2D2for5′-CAAACGTGGAACGACACTGTTGGCTGGATC-3′An2D2rev5′-TGCGTGATCTTCCACGAGAAATAGGGGAC-3′An2E2for5′-TTTCAGCTGGAAGATCTCTCACGGCAAG-3′An2E2rev5′-GCTAATAAGGTTGTAAGTGGCCAAGGGGGTCTC-3′An2F2for5′-GAGGACACGCCCGTCGCCACCCACTCCATCCTG-3′An2F2rev5′-GGCGCTAACGTAAAAGGTGAGCAGGTACAAGGG-3′An2G2for5′-CAGATGTATCTCTGTAACGTTACCGGTCACGACA-3′An2G2rev5′-CAGCAACCCCAGGTCGATATCACTCAGATTCATGA-3′An2H2for5′-CTGATTGCGATTTCGGGCCTCGCCATCACCGCC-3′An2H2rev5′-AGGGGTGTGTGTGCTGGAGGTAGGTGATGGC-3′An2I2for5′-CCACACACACCCCACTCTCCCCCCACTACCAGC-3′An2rev5′-GTCACTCGAGGTTAGGCTTGGTCAGCTT-3′ Open table in a new tab Expression in Saccharomyces cerevisiae—Transformation, selection, and growth of transgenic yeast cells were performed as described (25Hornung E. Korfei M. Pernstich C. Struss A. Kindl H. Fulda M. Feussner I. Biochim. Biophys. Acta. 2005; 1686: 181-189Crossref PubMed Scopus (27) Google Scholar). Expression of desaturases was induced by supplementing galactose (2%) to the media. If fatty acids were added (at a concentration of 0.02% (w/v)), the media was additionally supplemented with Igepal CA 630 (Nonidet P-40′) from Fluka (Sigma-Aldrich) at a concentration of 0.2% (w/v). Cultures were maintained at 16 °C for 10 days with shaking (140 rpm) to A600 densities of 10–20. Lipid analysis of transgenic yeast cells was performed after harvesting and lyophilization of 100-ml cultures supplemented with linoleic acid or without additional fatty acids. Absolute fatty acid accumulation varied with expression levels between experiments and with different expression vectors (data not shown) and, therefore, specific enzyme activities cannot be directly compared, allowing only for qualitative evaluation of altered enzyme specificities. Growth conditions of chimeric enzymes: Expression cultures were grown at 30 °C in minimal medium lacking uracil, but containing 2% (w/v) galactose. When these cultures had reached an OD600 of 0.5–0.8, they were transferred to room temperature (23–25 °C). After 96 h at 23–25 °C, cells were harvested by centrifugation at 1200 × g for 5 min, and pellets were washed once with sterile doubled-distilled H2O before being used for fatty acid analysis. The host strain transformed with the empty vector was used as a negative control in all experiments. Total and Esterified Fatty Acid Analysis—For the analysis of esterified fatty acids, 25 ml of sedimented and lyophilized yeast cultures were homogenized in derivatization solution. Fatty acids were transesterified with sodium methoxide in methanol or methanol/toluol solution (26Hornung E. Pernstich C. Feussner I. Eur. J. Biochem. 2002; 269: 4852-4859Crossref PubMed Scopus (78) Google Scholar). Fatty acid methyl esters (FAMEs) were then analyzed by gas-liquid chromatography. For the determination and verification of the position of double bond positions in fatty acids, fatty acid methyl esters were converted into their 4,4-dimethyloxazoline derivatives as described (27Christie W.W. Lipids. 1998; 33: 343-353Crossref PubMed Scopus (228) Google Scholar) and analyzed mass spectrometrically according to Ref. 27Christie W.W. Lipids. 1998; 33: 343-353Crossref PubMed Scopus (228) Google Scholar, using the 6890 Gas Chromatograph/5973 Mass Selective Detector system (Agilent). For the analysis of total fatty acids, FAMEs were prepared from sedimented cell pellets by direct transmethylation with methanol containing 2% (v/v) dimethoxypropane and 2.75% (v/v) of sulfuric acid. After 1 h at 80 °C, 0.2 ml of 5 m NaCl was added, and FAMEs were extracted with 2 ml of hexane. Lipid Analysis and Positional Analysis—Extraction and separation of phospholipids by TLC were performed as described (25Hornung E. Korfei M. Pernstich C. Struss A. Kindl H. Fulda M. Feussner I. Biochim. Biophys. Acta. 2005; 1686: 181-189Crossref PubMed Scopus (27) Google Scholar). After extraction of the phospholipids from the silica matrix 1/10 of each sample was transesterified with sodium methoxide and analyzed by GC. In the rest of the sample, the positional distribution of fatty acids was determined by dissolving the phospholipids in 900 μl of Tris-buffer (40 mm Tris-HCl, pH 7.2; 0.2% v/v Triton 100; 50 mm boric acid) by sonication. 100 units of Rhizopus arrhizus lipase (Fluka) were added, and the digestion conducted for 2 h at 37 °C. 100 μl of acetic acid was added to stop the reaction, and the aqueous phase was extracted with chloroform and methanol (28Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar). The resulting organic phase was dried under nitrogen stream and dissolved in 800 μl of methanol. 400 μl of each sample was transesterified with sodium methoxide to convert lysophospholipids to FAMEs. 6.5 μl of trimethylsilyldiazomethane was added to the rest of each sample to convert free fatty acids into their corresponding methyl esters, shaken for 30 min at room temperature, and 0.2 μl of acetic acid was added to degrade remaining trimethylsilyldiazomethane. Samples were dried under nitrogen stream. The resulting FAMEs were dissolved in acetonitrile and analyzed by GC. GC analysis was performed with an Agilent GC 6890 system coupled with an FID detector equipped with a capillary HP INNOWAX column (30 m × 0.32 mm; 0.5 μm coating thickness; Agilent, Germany). Helium was used as a carrier gas (1 ml min–1). Samples were injected at 220 °C. The temperature gradient was 150 °C for 1 min, 150–200 °C at 15 °C min–1, 200–250 °C at 2 °C min–1, and 250 °C for 10 min. Fatty acids were identified according to authentic standards. GC/MS analysis was carried out using an Agilent 5973 Network mass selective detector connected to an Agilent 6890 gas chromatograph equipped with a capillary DB-23 column (30 m × 0.25 mm; 0.25-μm coating thickness; J&W Scientific, Agilent. Helium was used as a carrier gas (1 ml min–1). Samples were injected at 220 °C. The temperature gradient was 150 °C for 1 min, 150-200 °C at 4 °C min–1, 200–250 °C at 5 °C min–1, and 250 °C for 10 min. The detector was set to an electron energy of 70 eV, an ion source temperature of 230 °C, and a transfer line temperature of 260 °C. Identification of Genes Encoding A. nidulans Membrane-bound Desaturases—To characterize and identify new desaturases from fungi, we searched the A. nidulans genome sequence (29Galagan J.E. Calvo S.E. Cuomo C. Ma L.J. Wortman J.R. Batzoglou S. Lee S.I. Basturkmen M. Spevak C.C. Clutterbuck J. Kapitonov V. Jurka J. Scazzocchio C. Farman M. Butler J. Purcell S. Harris S. Braus G.H. Draht O. Busch S. D'Enfert C. Bouchier C. Goldman G.H. Bell-Pedersen D. Griffiths-Jones S. Doonan J.H. Yu J. Vienken K. Pain A. Freitag M. Selker E.U. Archer D.B. Penalva M.A. Oakley B.R. Momany M. Tanaka T. Kumagai T. Asai K. Machida M. Nierman W.C. Denning D.W. Caddick M. Hynes M. Paoletti M. Fischer R. Miller B. Dyer P. Sachs M.S. Osmani S.A. Birren B.W. Nature. 2005; 438: 1105-1115Crossref PubMed Scopus (1042) Google Scholar) databases with known query desaturase sequences from plants. Two sequences with similarity to known plant desaturases were identified. The genomic sequence information was used to design primers to amplify putative fatty acid desaturases from cDNA prepared from A. nidulans cultures in vegetative and sexual growth states. Two cDNA fragments were successfully amplified, cloned, and sequenced. The resulting deduced full ORF of the first sequence, An2 (GenBank™ accession no. XP_658641), encodes a polypeptide of 426 amino acids and is largely identical with the annotated gene odeA (GenBank™ accession no. AF262955) with the exception of six additional encoded amino acids at the C terminus of An2. The gene odeA has been suggested to encode a fatty acid Δ12 desaturase according to knock-out studies in A. nidulans (21Calvo A.M. Gardner H.W. Keller N.P. J. Biol. Chem. 2001; 276: 25766-25774Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The second sequence identified, An1 (GenBank™ accession no. XP_664808) encodes a polypeptide of 394 amino acids. Sequencing of the cloned cDNA fragments for An1 revealed An1 to be longer than the predicted and published sequence from the genome data base because of insertion of eight amino acids following amino acid position 13. The longer sequence has been delineated before and associated with a putative fatty acid ω3-desaturase in a patent (30Ursin V.M. Voelker T. Froman B. PCT WO 03/099216 A2. 2003; Google Scholar). Classification of An2 and An1 Sequences in the Phylogenetic System—Pairs of Δ12 desaturase-like proteins similar to that found in A. nidulans have been reported from several fungal species, and the relationship between these fungal enzymes has led Damude et al. (19Damude H.G. Zhang H. Farrall L. Ripp K.G. Tomb J-F. Hollerbach D. Yadav N.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9446-9451Crossref PubMed Scopus (136) Google Scholar) to establish two subfamilies for monofunctional and bifunctional enzymes, respectively; hence the description of An2 and An1. The polypeptides deduced from the An2 and An1 cDNA sequences share 45% identity. The deduced An2 protein is most similar to representatives of the group of monofunctional desaturases (19Damude H.G. Zhang H. Farrall L. Ripp K.G. Tomb J-F. Hollerbach D. Yadav N.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9446-9451Crossref PubMed Scopus (136) Google Scholar), with the highest degree of similarity to the putative Δ12 desaturase from Neurospora crassa (GenBank™ accession no. XP_959528, 70% identity), followed by the putative Δ12 desaturases from M. grisea (GenBank™ accession no. XP_365283, 69% identity) and from F. moniliforme (GenBank™ accession no. ABB88515, 63% identity). Similarity of the deduced An2 protein toward bifunctional enzymes from the species mentioned above is around 45% identity. The situation for the deduced An1 protein from A. nidulans is reversed in that with ∼60% identity in all cases, the An1 sequence is most similar to representatives of the bifunctional desaturase group (19Damude H.G. Zhan