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:
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.

This work was supported by the BASF Plant Science GmbH.
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