Báo cáo Y học: Formation of conjugated D11 D13-double bonds by D12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds doc

Formation of conjugated D 11 D 13 -double bonds by D 12 -linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds Ellen Hornung 1 , Christian Pernstich 1 and Ivo Feussner 1,2 1 Institut fu ¨ r Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany; 2 Albrecht-von-Haller-Institut fu ¨ r Pflanzenwissenschaften, Georg-August Universita ¨ t Goettingen, D-37077 Goettingen, Germany For the biosynthesis of punicic acid (18:3 D9Z,11E,13Z )a (11,14)-linoleoyl desaturase activity has been proposed. To isolate this acyl-lipid-desaturase, PCR-based cloning was used. This approach resulted in the isolation of two complete cDNAs. The first isolated full-length cDNA harbors a sequence of 1350 bp encoding a protein of 395 amino acids. The second cDNA was 1415 bp long encoding a protein of 387 amino acids. For functional identification proteins encoded by the cDNAs were expressed in Saccharomyces cerevisiae, and formation of newly formed fatty acids was analyzed by gas chromatography-free induction decay (GC-FID) and GC/MS. The expression of the heterologous enzymes resulted in the first case in a significant amount of linoleic acid and in the second case, after linoleic acid sup- plementation, in formation of punicic acid. The results presented here identify one cDNA coding for a classical D 12 -acyl-lipid-desaturase. The other one codes for a new type of (1,4)-acyl-lipid-desaturase that converts a cis double bond located in the D 12 -position of linoleic acid or c-linolenic acid, but not in a-linolenic acid, into a conjugated cis–trans double bond system. Keywords: acyl-group desaturase; conjugase; Punica granatum; Saccharomyces cerevisiae; seed oil. The most common octadecatrienoic fatty acid in plants is a-linoleic acid (18:3 D9Z,12Z,15Z ), which is the main constitu- ent of chloroplastic membranes [1]. Beside this, triacylglyce- rols from seeds are sometimes composed of additional conjugated octadecatrienoic acids having (Z,E,E)or (Z,E,Z) geometries [2], providing an easily accessible source of these fatty acids. At least five different out of the six theoretical invisible regio-isomers have been reported within plant seed oils with double bond systems in the following positions: (Z,E,Z)- and (E,E,Z)-8,10,12–18:3 and (Z,E,Z)- (Z,E,E)- and (E,E,Z)-9,11,13–18:3. One of these, punicic acid (18:3 D9Z,11E,13Z ) is the major constituent of the seed oil of Punica granatum [3]. Seed oils harboring conjugated fatty acids are of industrial interest, because the oil is used as drying oil in paints and may be used for cosmetic purposes. A number of enzymatic mechanisms have been published to describe the biosynthesis of conjugated octadecatrienoic acids in plants. These include an oxidase type reaction [4] and the direct isomerization of linolenic acid [5,6] at the level of free fatty acids in algae. In recent publications on the biosynthesis of a-eleostearic acid (18:3 D9Z,11E,13E ) and of calendic acid (18:3 D8E,10E,12Z ) [7–10], it became clear that the responsible enzymes in higher plants belong to the growing family of special acyl-lipid-desaturases (Fig. 1) [11,12]. Besides introducing conjugated double bonds by so-called (1,4)-acyl-lipid-desaturases (FADX) this class of enzymes catalyzes the formation of hydroxy, epoxy, and acetylenic groups, respectively, within a fatty acid backbone [13,14]. Furthermore the reaction takes place while the acyl moiety is esterified to PtdCho as has been shown first for the classical acyl-lipid-desaturases [15] and then for the forma- tion of a-eleostearic acid as well [16]. However, all (1,4)-acyl- lipid-desaturases isolated so far from plants convert a cis double bond either at position D 9 or D 12 , respectively, of the fatty acid backbone into a conjugated trans–trans double bond system. To obtain additional information on the biosynthesis of conjugated octadecatrienoic acids we deci- ded to expand the analysis on the biosynthesis of punicic acid (18:3 D9Z,11E,13Z ) in the seeds of P. granatum,since the biosynthesis of this conjutrienoic fatty acid involves the conversion of a cis double bond at position D 12 into a conjugated trans–cis double bond system. Here, we describe the cloning of this new type of (1,4)-acyl-lipid-desaturase that catalyzes the formation of a conjugated triene fatty acid that harbors a (Z,E,Z)-9,11,13–18:3 double bond system. MATERIALS AND METHODS Chemicals Standards of fatty acids as well as all other chemicals were from Sigma (Deisenhofen, Germany). Conjugated fatty acids were from Larodan (Malmo ¨ , Sweden), methanol, hexane and 2-propanol (all HPLC grade) were from Baker (Deventer, the Netherlands). Correspondence to I. Feussner, Biochemie der Pflanze, AvH, Justus-von-Liebig-Weg 11, D-37077 Goettingen, Germany. Fax: 49 551 395749, Tel.: + 49 551 395743, E-mail: ifeussn@gwdg.de Abbreviations: FAD12, D 12 -fatty acid desaturase; FADX, (1,4)-acyl- lipid desaturase; FAD-OH, fatty acid hydroxylase; GC-FID, gas chromatography-free induction decay; PCI, phenol/chloroform/ isoamyl alcohol; PVP, polyvinylpyrrolidone. Note: The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL data bank with accession numbers PuFAD12 AJ437139, PuFADX AJ437140. (Received 28 May 2002, revised 3 August 2002, accepted 15 August 2002) Eur. J. Biochem. 269, 4852–4859 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03184.x Isolation of cDNAs P. granatum seeds were harvested from fruits obtained from a local market. For RNA isolation, 20 g of seeds were ground in liquid nitrogen, 200 mL of extraction buffer I [100 m M Tris/HCl, pH 7.5, 25 m M EDTA, 2% (w/v) laurylsarcosyl, 4 M guanidinium thiocyanate, 5% (w/v) polyvinylpyrrolidone (PVP), 1% (v/v) a-mercapto- ethanol] was added, homogenized further with an Ultra- turrax (IKA Labortechnik, Staufen, Germany) and the homogenate was shaken for 10 min. After centrifugation at 3000 g for 15 min the floating solid lipid phase and the pellet were discarded, and the remaining liquid phase was extracted with an equal volume of PCI (phenol/chloroform/ isoamyl alcohol, 20 : 20 : 1, v/v/v). After centrifugation at 3000 g for 10 min the hydrophilic phase was reextracted with an equal volume of chloroform and the centrifugation step performed as before. The hydrophilic phase was loaded on a CsCl cushion (5 M CsCl) of 8 mL and centrifuged at 18 °C and 100 000 g for 18 h. The RNA precipitate was dried, resuspended and extracted for 15 min in a mixture consisting of 7.5 mL extraction buffer II (100 m M Tris/HCl, pH 8.8, 100 m M NaCl, 5 m M EDTA, 2% (w/v) SDS and 10 mL PCI). Centrifugation and washing of the hydrophilic phase with chloroform followed and the RNA was preci- pitated from the hydrophilic phase by an equal volume of 5 M LiCl overnight at 4 °C. After centrifugation for 30 min at 120 000 g at 4 °C the precipitate was washed with 70% ethanol, dried and dissolved in 1 mL water. From this total RNA fraction poly(A) + RNA was enriched using the Poly Attract-Kit TM (Promega, Mannheim, Germany) according to the manual provided, and used for all further experi- ments. ss-cDNA was synthesized from poly(A) + RNA of pomegranate seeds by reversed transcription with Super- scriptII TM (Gibco BRL, Eggenstein, Germany). Construction of expression plasmids and recombinant protein synthesis This ss-cDNA was used as template for PCR-based cloning. PCR fragments of about 560 bp were amplified with the degenerate sense primer A 5¢-TGGGTIAWHGCHCAYG ARTGBGG-3¢ and antisense primer B 5¢-CCARTYCCAY TCIGWBGARTCRTARTG-3¢, derived from the amino acid sequences WVIAHEC and HYDS(S/T)EW(D/N)W, respectively which in acyl-lipid-desaturases are highly con- served. PCR was carried out with TfI-DNA-Polymerase TM (Biozym, Hess. Oldendorf, Germany) using an amplification program of 2 min denaturation at 94 °C, followed by 10 cycles of 30 s at 94 °C, 45 s at 50 °C, 1 min at 72 °C, followed by 20 cycles of 30 s at 94 °C, 45 s at 50 °C, 1 min at 72 °C (time increment 5 s) and terminated by 2 min extension at 72 °C. PCR products of the expected length wereclonedinpGEM-T TM (Promega, Mannheim, Germany) and sequenced. The fragments Pun1 and Pun2 were chosen for the isolation of full-length cDNA clones using the marathon TM cDNA amplification kit (Clontech, Heidelberg, Germany). To amplify the 5¢-and3¢-ends of Pun1 and Pun2 by PCR, specific primers were used. 5¢-RACE primer C: 5¢-GGG ACG AGG AGC GAT GTG TGG AG-3¢, Pun1 3¢-RACE primer D: 5¢-AGT CCT CAT ATT AAA TGC ATT CGT GG-3¢, Pun2 5¢-RACE primer F: 5¢-ACG GAA CGA GGA GCG CTG AGT G-3¢,3¢-RACE primer G: 5¢-CTG ATC GTG AAC GCA TTC CTG G-3¢. Amplification was carried out by using the Advantage cDNA PCR-Kit TM (Clontech, Hei- delberg, Germany) according to the manufacturer’s instruc- tions. The fragments were cloned in pGEM-T TM and sequenced. To obtain the full-length cDNA clones by PCR and for expression in S. cerevisiae, specific primers of the expected open reading frame of the entire cDNA with suitable recognition sites were used for amplification. Pun1 sense primer H 5¢-ATG GGA GCT GAT GGA ACA ATG TCT C-3¢, antisense primer I 5¢-ATT CAG AAC TTG CTC TTG AAC CAT AG-3¢ and Pun2 sense primer J5¢-ATG GGA GCC GGT GGA AGA ATG AC-3¢ anti- sense primer K 5¢-TGA TCA GAG GTT CTT CTT GTA CCA G-3¢. The Expand TM High Fidelity-System (Roche Diagnostics, Mannheim, Germany) was used, with an amplification program of 2 min denaturation at 94 °C, followed by 10 cycles of 30 s at 94 °C, 30 s at 58 °C, 1 min at 72 °C, followed by 15 cycles of 30 s at 94 °C, 30 s at 58 °C, 1 min at 72 °C (time increment 5 s) and terminated by 5 min extension at 72 °C. The fragments were cloned into pGEM-T TM and the resulting plasmids PuFADX and PuFAD12 were sequenced. For expression in S. cerevisiae the open reading frames of PuFADX and PuFAD12 were cloned as a HindIII/BamHI or SalI/HindIII fragment, respectively, behind the galactose-inducible promotor GAL1 into the shuttle vector pYES2 TM (Invitrogen, Carls- bad, USA) or pESC-LEU TM (Stratagene, Amsterdam, the Netherlands) to yield the plasmids pYES-PuFADX and pESC-LEU-PuFAD12. The plasmids were transformed into the yeast strain INVSc1 TM (Invitrogen, Carlsbad, USA) by lithium acetate [17]. Individual colonies of cell were then grown overnight at 30 °C in SD media lacking uracil (pYES2 TM ) or leucin (pESC-LEU TM ), supplemented with glucose. Cells were then washed twice in SD media, before being diluted to A 600 ¼ 0.2–0.4 in SD media supplemented with galactose. If fatty acids were added, at a concentration of 0.02% (w/v), the media was also supplemented with tergitol type NP-40 at a concentration of 0.2% (w/v). Cultures were maintained either for 3 days at 30 °C or for 10 days at 16 °C with shaking (150 r.p.m) to densities of A 600 ¼ 2–3. Twenty millilitres of cell cultures were harvested by centrifugation and lyophilized. Lipid analysis For analysis of punicic acid content of transformed yeast cells, lyophilized cell pellets were homogenized by adding Fig. 1. Reactions catalyzed by members of the acyl-lipid-desaturase family. Ó FEBS 2002 Punicic acid producing (1,4)-acyl-lipid-desaturase (Eur. J. Biochem. 269) 4853 1.35 mL of a mixture of toluene and methanol (1 : 2, v/v) and 0.5 mL sodium methoxide, using a glass rod. After shaking the samples for 20 min at room temperature, 1.8 mL of 1 M NaCl and 4 mL heptane were added and fatty acid methyl esters were extracted by shaking vigor- ously for 10 min. The organic phase was evaporated to dryness under a nitrogen stream and the corresponding fatty acid methyl esters were reconstituted in 40 lLof acetonitrile. Then 1 lL of each sample was analyzed by GC, performed with an Agilent GC 6890 system (Agilent, Waldbronn, Germany) coupled with an FID detector equipped with a capillary HP INNOWAX column (30 m · 0.32 mm, 0.5 lm coating thickness, Agilent, Wald- bronn, Germany). Helium was used as the carrier gas (30 cm · s )1 ). The samples were measured with a split of 20 : 1 with an injector temperature of 220 °C. The tem- perature gradient was 150 °C for 1 min, 150–200 °Cat 15 °Cmin )1 , 200–250 °Cat2°Cmin )1 ,and250°Cfor 10 min. Fatty acids were identified by authentic standards. Alternatively, the corresponding fatty acid methyl esters were analyzed by GC/MS, performed with an Agilent GC 6890 system coupled with an Agilent 5973 N MS detector (Agilent, Waldbronn, Germany). The GC was equipped with a capillary HP-5 column (5% diphenyl : 95% polydi- methyl siloxane, 30 m · 0.25 mm, 0.25 lm coating thick- ness, Agilent, Waldbronn, Germany) and helium was used as the carrier gas (40 cm · s )1 ). An electron energy of 70 eV, an ion source temperature of 230 °C, and a temperature of 275 °C for the transfer line were used. The samples were measured in the EI mode, and the splitless injection mode (opened after 1 min) with an injector temperature of 250 °C. The temperature gradient was 60– 110 °Cat25°CÆmin )1 , 110 °C for 1 min, 110–270 °Cat 10 °CÆmin )1 , and 270 °C for 10 min. RESULTS PCR-based cloning and isolation of full-length cDNAs coding for acyl-lipid-desaturases For PCR-based cloning degenerated primers, deduced from conserved regions of acyl-lipid-desaturases, were synthes- ized [9]. The template used was ss-cDNA from P. granatum, which was reverse-transcribed from mRNA of seeds from fruits. PCR products of the expected length were cloned and sequenced. Database searches and alignments with these fragments indicated two different fragments (Pun1 and Pun2) with similarities to plant acyl-lipid-desaturases. Pun1 was a fragment of 586 bp. The corresponding amino acid sequence exhibited highest identities to D 12 -fatty acid desaturases from Gossypium hirsutum (accession number Y10112) and Solanum commersonii (accession number X92847). The corresponding amino acid sequence of Pun2, a fragment of 567 bp, showed highest identities to D 12 -fatty acid desaturases from Sesamum indicum (accession number AF192486) and again to a D 12 -fatty acid desaturase from S. commersonii. To isolate the full-length cDNA clones, RACE with specific primers was used to amplify the 5¢-and3¢-ends of Pun1 and Pun2. The fragments were cloned and sequenced. With specific primers for the expected open reading frames containing specific restric- tion sites the entire cDNAs of about 1.2 kb were amplified by PCR and subcloned into pGEM-T TM . The resulting fragments were sequenced. The full-length cDNA of Pun1 had a length of 1185 bp coding for a protein of 395 amino acids with a calculated molecular mass of 45.8 kDa. The amino acid sequence of this putative fatty acid desaturase showed highest identities to the D 12 -fatty acid desaturases from G. hirsutum (58%), from S. commersonii (59%) and from Corylus avellana (61%, accession number A65100), respectively. A more detailed comparison of these sequences is shown in Fig. 2. Due the low sequence identity of the encoded protein against classical D 12 -fatty acid desaturases it was expected that this clone may code for a (1,4)-acyl- lipid-desaturase. It was therefore named PuFADX. The full-length cDNA of Pun2 had a length of 1161 bp coding for a protein of 387 amino acids with a calculated molecular mass of 44.3 kDa. The corresponding amino acid sequence showed higher identities against those of the classical D 12 - fatty acid desaturases from S. indicum (78%), S. commerso- nii (77%) and C. avellana (78%), respectively, and was therefore named PuFAD12 (Fig. 2). These findings were substantiated further by phylogenetic tree analysis Fig. 3. This analysis indicates that PuFADX, similar to all other (1,4)-acyl-lipid-desaturases isolated so far which modify a double bond at position D 12 of linoleic acid, groups into one subgroup of the acyl-lipid-desaturase family. There is only one exception that is the (1,4)-acyl-lipid-desaturase from Impatiens, which in contrast modifies a-linolenic acid preferentially. D 12 -acyl-lipid-acetylenases and epoxygenases as well as D 9 -(1,4)-acyl-lipid-desaturases form another subgroup within this phylogenetic tree. Functional expression in S. cerevisiae and fatty acid analysis To investigate the product and substrate specificity of PuFAD12 and PuFADX, respectively, the full-length cDNAs were cloned into yeast expression vectors under the control of the inducible GAL1 promoter and the encoded proteins were expressed in S. cerevisiae strain INVSc1. In induced cultures of cells harboring the cDNA of PuFAD12 accumulation of linoleic acid and to a much lower extent of hexadecadienoic acid was observed (Fig. 4, upper panel vs. middle panel). The accumulation was dependent on the growth temperature of the cultures as little or no linoleic acid and hexadecadienoic acid were detected in cells maintained at 30 °C. Whereas linoleic acid and hexadecadienoic acid accumulated up to 5% and 1% (w/w), respectively, of the total fatty acids, if cells were grown at 16 °C. Since PuFADX was expected to code for a (1,4)-acyl- lipid-desaturase, cultures transformed with PuFADX were supplemented with linoleic acid as putative substrate. However in induced yeast cultures transformed with PuFADX and without the addition of linoleic acid to the growth medium, accumulation of linoleic acid up to 1.2% (w/w) has been observed, if the cells were maintained at 30 °C (data not shown). Punicic acid could only be detected after supplementation of the growth media with linoleic acid thus confirming again this fatty acid as the precursor of plant (1,4)-acyl-lipid-desaturases producing trienoic fatty acids (Fig. 5, upper panel vs. middle panel). The accumu- lation of punicic acid was reduced at lower temperatures. This was in contrast to the increased accumulation of linoleic acid and hexadecadienoic acid in cells expressing 4854 E. Hornung et al.(Eur. J. Biochem. 269) Ó FEBS 2002 PuFAD12 at this temperature. In cells maintained at 30 °C and supplemented with linoleic acid, punicic acid accounted for up to 1.6% (w/w) of the total fatty acids. The identity of the conjutrienoic fatty acid methyl ester punicic acid in yeast cell cultures was established by gas chromatographic retention times of the methyl esters of three different positional isomers as authentic standards. In the lower panel of Fig. 5 it is shown that the different positional isomers of conjutrienoic fatty acids could be clearly separated under the conditions used. In addition, mass spectrometry was performed to confirm the identity of the substance assigned as punicic acid. The mass spectrum of this fatty acid methyl ester, shown in the upper panel of Fig. 6, was identical to that of methyl punicic acid and was characterized by an abundant molecular ion of m/z ¼ 292 which has been shown before to be characteristic for conjugated fatty acids [7]. To investigate further the substrate specificity of PuFADX, induced yeast cultures were supplemented either with cis-ortrans-vaccenic acid, a-orc-linolenic acid or with homo-c-linolenic acid, respectively, and grown at 30 °C. With cis-andtrans-vaccenic acid, a-linolenic acid and homo-c-linolenic acid no formation of a conjugated fatty acid was found (data not shown and Table 1). However with c-linolenic acid the formation of a presumably conjugated octatetraenoic fatty acid was found (Table 1). In order to confirm the structure of this newly formed fatty acid, mass spectrometry was used and in the lower panel of Fig. 6 the resulting mass spectrum is shown. Again the fatty acid methyl ester was characterized by an abundant molecular ion that time of m/z ¼ 290, thus confirming the Fig. 2. Sequence alignment of the D 12 -acyl-lipid-desaturases. The pro- tein alignment was generated with the CLUSTAL - X program and was performed with sequences from S. indicum (SiFAD12, accession number AF192486), S. commersonii (ScFAD12, accession number X92847), P. granatum (PuFAD12, accession number AJ437139), G.hirsutum (GhFAD12, accession number Y10112), Crepis alpina (CaFAD12, accession number Y16285), and the D 12 -acyl-lipid- desaturase from P. granatum (PuFADX, accession number AJ437140). Boxes indicate the three characteristic and highly conserved histidine regions and identical amino acids are marked as bold letters. For the alignment D 12 -acyl lipid desaturases were selected which displayed the highest amino acid identities towards the two newly described D 12 -acyl- lipid-desaturases from pomegranate. Fig. 3. Phylogenetic tree analysis of plant acyl-lipid-desaturases. The protein alignment was generated with the CLUSTAL - X program, and the phylogenetic tree was made with TREEVIEW . Arabidopsis thaliana: AtFAD12 (ATD12aaa); Calendula officinalis: CoFAD12 (AF343065), CoFADX-1 (AF310155), CoFADX-2 (AF310156); Crepis alpina: CaFAD12ace (Y16285); Crepis palaestina: CpFAD12 (Y16284); CpFAD12epo (Y16283); Daucus carota: DcFAD12-OH (AF349965); Dimorphotheca sinuata: DmFAD12t (WO 01/128000), DmFADX-OH (WO 01/128000); G. hirsutum: GhFAD12 (Y10112); Glycine max: GmFAD12 (L43921); Helianthus annuus: HaFAD12 (AF251842); I. balsamina: IbFADX (AF182520); Lesquerella fendleri:LfFAD12- OH (AF016103); Licania michauxii: LmFADX (WO 00/11176); Momordica charantia; McFADX (AF182521); P. granatum; PuFAD12 (AJ437139), PuFADX (AJ437140); Ricinus communis; RcFAD12-OH (U23378); S. commersonii: ScFAD12 (X92847); S. indicum: SiFAD12 (AF192486); Vernonia galamensis: VgFAD12-1 (AF188263), VgFAD12-2 (AF188264). FAD12: D 12 -fatty acid desaturase, FADX: (1,4)-acyl-lipid-desaturase, FAD-OH: fatty acid hydroxylase. Ó FEBS 2002 Punicic acid producing (1,4)-acyl-lipid-desaturase (Eur. J. Biochem. 269) 4855 formation of a conjugated fatty acid. To compare the substrate specificity directly between linoleic acid and c-linolenic acid, yeast cells harboring PuFADX were grown in the presence of an equimolar mixture of linoleic acid, a-linolenic acid and c-linolenic acid. The resulting fatty acid profile is shown in the middle panel of Fig. 7. Punicic acid and the conjugated octadecatetraenoic fatty acid derived from c-linolenic acid but not from a-linolenic acid were detected in a ratio of 2 to 0.6% (w/w) indicating a three- to fourfold preference of PuFADX against linoleic acid under these conditions. DISCUSSION Over the last five years more and more data have accumulated which show that the growing family of special acyl-lipid-desaturases catalyzes the formation of a wide array of functional groups within unusual fatty acids predominantly found in plant seed oils [18]. To this family belong now besides the classical D 12 and D 15 -acyl-lipid- desaturases [12], desaturases directly fused to their elec- tron donor such as D 5 and D 6 -acyl-lipid-desaturases [19], hydroxylases [14], acetylenases and epoxygenases [13], (1,4)- acyl-lipid-desaturases [7,10], and recently desaturases which form hydroxy groups in conjugation to double bonds [20]. Here, we report on the isolation of two diverse acyl-lipid- desaturases, PuFAD12 and PuFADX, respectively, from pomegranate seeds. Both cDNAs have sequence similarity to acyl-lipid-desaturases from plants. PuFAD12 has higher amino acid identity to the classical D 12 -acyl-lipid-desaturases (approximately 80%), whereas PuFADX has equal Fig. 4. GC/FID analysis of fatty acid methyl esters isolated from yeast cells transformed with pESC-LEU-PuFAD12. The lipids were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and analyzed by GC as described under materials and methods. The upper panel shows the fatty acid profile of nontransformed yeast cells as controls. All fatty acids were characterized by coelution of authentic standards and the lower panel shows a linoleic acid standard. Fig. 5. GC/FID analysis of fatty acid methyl esters isolated from yeast cells supplemented with linoleic acid and transformed with pYES- PuFADX. The lipids were extracted from lyophilized yeast cells, este- rified fatty acids were transmethylated and analyzed by GC as described in Materials and methods. The upper panel shows the fatty acid profile of nontransformed yeast cells as controls. All fatty acids were characterized by coelution of authentic standards and the lower panel shows a standard mixture of three different conjutrienoic fatty acids. 4856 E. Hornung et al.(Eur. J. Biochem. 269) Ó FEBS 2002 sequence identity (approximately 60%) to both classical D 12 - desaturases and D 12 -desaturase-related nonclassical acyl- lipid-desaturases. Expression of PuFAD12 in yeast cells indicated that it is a classical D 12 -acyl-lipid-desaturase (Fig. 4). Expression of PuFADX in yeast cells revealed that the enzyme produced fatty acid derivatives with conjugated double bond systems from linoleic and c-linolenic acid substrates, respectively (Fig. 5, Table 1). Two names, ÔconjugaseÕ and Ô(1,4)-acyl-lipid-desaturaseÕ were previously suggested to refer to enzymes that are responsible for introducing conjugated double bonds into acyl chains [7,9]. Both names were proposed, because they describe the catalytic mechanism of these enzymes: ÔConju- gaseÕ, since the enzyme forms two conjugated double bonds out of one isolated double bond and Ô(1,4)-acyl lipid desaturaseÕ, since this class of enzymes seem to catalyze an (1,4)-elimination of hydrogen atoms bound to the aliphatic carbon chain instead of an 1,2-syn elimination in case of the classical acyl-lipid-desaturases [21]. Two (1,4)-acyl-lipid- desaturases from Impatiens balsamina and Momordica cha- rantia were found to be able to convert the D 12 -double bond of linoleic acid into two conjugated and trans configurated double bonds at the 11 and 13 positions, resulting in the production of the conjugated linolenic acid 18:3 D9Z,11E,13E [7]. In addition (1,4)-acyl-lipid-desaturases isolated from C. officinalis have been shown to convert the D 9 -double bond of linoleic acid again into two conjugated and trans configurated double bonds at the 8 and 10 positions to produce another conjugated linolenic acid derivative 18:3 D8E,10E,12Z [8–10]. As shown here by heterologous expression in yeast cells the enzyme PuFADX converts linoleic acid into another conjugated linolenic acid deri- vative (Fig. 5). In contrast to all other yet known (1,4)-acyl- lipid-desaturases, this enzymes converts the D 12 -double bond of linoleic acid into two conjugated and trans–cis configurated double bonds at the 11 and 13 positions, resulting in the production of the conjugated linolenic acid 18:3 D9Z,11E,13Z . It will be interesting to see by which mechani- stic parameters within the different enzymes the formation of either a (Z,E,E)or(Z,E,Z)-configurated double bond system of the different linolenic acid isomers is determined. Plant oils containing conjugated linolenic acid derivatives are of commercial interest, since they are used as drying oils in paints. Thus seed oils may be useful to be produced via transgenic approaches in a commercially important crop. This seed oil must contain significant amounts of the envis- aged product in a chemically pure manner. The amount of an unusual fatty acid is determined by (a) the complex Fig. 6. Mass spectra of conjugated fatty acid methyl esters. Conjugated fatty acid methyl esters were isolated from yeast cells transformed with PuFADX and supplemented either with linoleic acid (upper panel) or c-linolenic acid (lower panel), respectively. The lipids were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and analyzed by GC/MS as described under materials and methods. All fatty acids were characterized by coelution of authentic standards. The mass spectra of the substances eluting at the retention times of the conjugated fatty acid methyl esters were recorded. Table 1. Substrate specificity of PuFADX. Yeast cells transformed with PuFADX were grown in the presence of different fatty acids. The lipids were extracted from lyophilized yeast cells, esterified fatty acids were transmethylated and GC/FID analysis of fatty acid methyl esters isolated from these yeast cultures was performed as described under materials and methods. All fatty acids were characterized by coelution of authentic standards. Fatty acid detected Supplemented fatty acid (%) a 18:2 D9Z,12Z 18:3 D6Z,9Z,12Z 18:3 D9Z,12Z,15Z 20:3 D8Z,11Z,14Z 16:0 15.0 19.0 12.9 16.0 16:1 D9Z 14.9 9.2 28.9 30.0 18:0 5.7 8.5 6.2 5.6 18:1 D9Z 12.3 9.1 29.3 26.4 16:2 D9Z,12Z b – – – 0.8 18:2 D9Z,12Z 48.4 – – 0.4 c 18:3 D9Z,11E,13Z b 1.6 – – – 18:3 D6Z,9Z,12Z – 53.7 – – Conjugated 18:4 – 0.5 – – 18:3 D9Z,12Z,15Z – – 22.1 – 20:3 D8Z,11Z,14Z – – – 20.3 a Amount of each fatty acid was expressed as relative ratio of all fatty acids found. b New detected fatty acids. c This fatty acid may be derived due to substrate impurities. Ó FEBS 2002 Punicic acid producing (1,4)-acyl-lipid-desaturase (Eur. J. Biochem. 269) 4857 biosynthetic pathway of unusual fatty acids in seeds [18], since many of these functional groups are introduced into the fatty acid backbone while the fatty acid is esterified to a molecule of PtdCho [15,16], and (b) by the specificity of the respective enzyme which introduces this functional group. Since this class of enzymes needs linoleic acid as substrate, it needs a 18:2-platform to fulfill its function. With that respect oil crop plants are needed which harbor high amounts of linoleic acid within their seed oils such as soybean, flax or sunflower [3]. However, their oils contain substantial amounts of a-linolenic acid and all (1,4)-acyl-lipid-desatu- rases reported so far showed no preference against linoleic acid in the presence of a-linolenic acid. This problem may be solved by using this new type of (1,4)-acyl-lipid-desaturase that converts a double bond located only in the D 12 -position of linoleic acid or c-linolenic acid, but not in a-linolenic acid, into a conjugated double bond system. Therefore this enzyme may have advantages over the previously known enzymes, since c-linolenic acid is not found in the seed oils of most crop plants. ACKNOWLEDGEMENTS The authors are grateful to M. Pu ¨ rschel for expert technical assistance. 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