Propertiesoftwomultifunctionalplantfatty acid
acetylenase/desaturase enzymes
Anders S. Carlsson
1
, Stefan Thomaeus
1
, Mats Hamberg
2
and Sten Stymne
1
1
Department of Crop Science, Swedish University of Agricultural Sciences, Alnarp, Sweden;
2
Department of Medical Biochemistry and
Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden
The propertiesof the D6 desaturase/acetyle nase from the
moss Ceratodon purpureus and the D12 acetylenase from the
dicot Crepis alpina were studied by e xpressing t he encoding
genes in Arabidopsis thaliana and Saccharomyces cerevisiae.
The acetylenase from C. alpina D12 desaturated both oleate
and linoleate with about equal efficiency. The d esaturation
of oleate gave rise to 9(Z),12(E)- and 9(Z),12(Z)-octadeca-
dienoates in a ratio of approximately 3 : 1. Experiments
using stereospecifically deuterated oleates showed that the
pro-R hydrogen atoms were removed from C-12 and C-13
in the introduction of the 12(Z) double bond , whereas the
pro-R and pro-S hydrogen atoms were removed from these
carbons during th e formation of t he 12( E) double bond. The
results suggested that the D12 acetylenase could accommo-
date oleate having either a cisoid or transoid conformation
of the C
12
-C
13
single bond, and t hat these conformers served
as precursors of the 12(Z)and12(E) double bonds,
respectively. However, only the 9(Z),12(Z)-octadecad ieno-
ate isomer could be further d esaturated to 9(Z)-octadecen-
12-ynoate (crepenynate) by the enzyme. The evolutionarily
closely related D12 epoxygenase from Crepis palaestina
had only weak desaturase activity but could also produce
9(Z),12(E)-octadecadienoate from oleate. The D6 acetyle-
nase/desaturase from C. purpureus, on the other hand,
produced only the 6(Z) isomers using C16 and C18 ac yl
groups possessing a D9 double bond as substrates. T he D6
double bond was efficiently further con verted to an acety-
lenic bond by a second round of desaturation but only if the
acyl substrate had a D12 double bond and that this was in
the Z configuration.
Keywords:acetylenase;Ceratodon purpureus; Crepis sp.;
desaturase; Saccharomyces cerevisiae.
Recently, a number ofplant genes have been cloned that
encode enzymes evolutionarily related to the D12 desatu-
rase converting oleate to linoleate [9(Z),12(Z)-octadeca-
dienoate]. These enzymes catalyze not only D12-cis
desaturation but also hydroxylation [1,2], epoxidation
[3], formation of acetylenic bonds [3,4] of conjugated
double bonds [5–8] and of E double bonds [9]. The
hydroxylases have been shown to also carry out D12(Z)
desaturation [2] and conjugases to be able to efficiently
D12 (E) desaturate oleate [7]. Here we report on the
multifunctionality a nd stereochemistry o f another mem-
ber of the D12 desaturase-like enzyme family, the Crepis
alpina D12 acetylenase, and show that this enzyme,
beside its ability to form triple bonds, is able to convert
oleic acid into a mixture of 12(E)and12(Z) isomers of
18:2. The catalytic activity of the C. alpina acetylen ase
enzyme is compared with that of the evolutionarily
closely related Crepis palaestina epoxygenase and another,
evolutionarily distantly related, bifunctional acetylenase/
desaturase from the moss Ceratodon purpureus.
Materials and methods
Plant material and growth conditions
Nontransformed Arabidopsis tha liana L. (Heynh) plants of
ecotype Columbia (wild type), its fad2 mutant defective
in the endoplasmatic reticular oleoyl D12 desaturase, as well
as transgenic A. thaliana, were grown in controlled growth
chamber at a photosynthetic flux of 100–120 lEÆm
)2
Æs
)1
,
20 °C in a photoperiod of 16 h light/8 h dark. C. palaestina
and C. alpina plants were grown under greenhouse condi-
tions.
Expression of D12 desaturase-like enzymes
in
A. thaliana
The cloning of the C. alpina D12 acetylenase (CREP1) as
well as the C. palaestina D12 epoxygenase ( CPAL2) genes
has been described earlier [3]. Full-length cDNA of CREP1
and CPAL2 were inserted into the binary vector o f pBI121
under the transcriptional c ontrol of a truncated version
(FP1) of the seed-specific napin promoter [10]. Transfor-
mation and cultivation of the A. thaliana plants was as
described earlier [11]. All fattyacid analyses of the
transformants were performed on T
3
seeds.
Correspondence to A. S. Carlsson, Department of Crop Science,
Swedish University of Agricultural Sciences, PO Box 44,
230 53 Alnarp, Sweden. Fax: + 46 40 415519, Tel.: + 46 40 415561,
E-mail: anders.carlsson@vv.slu.se
Abbreviations: 18:1D9(Z), oleic acid; 18:2D9(Z),12(Z), linoleic acid;
18:2D9(Z),12a, crepenynic acid.
Note: a web page is available at http://www.vv.slu.se
(Received 2 April 2004, revised 21 May 2004, accepted 26 May 2004)
Eur. J. Biochem. 271, 2991–2997 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04231.x
Expression of D6- and D12-desaturase-related enzymes
in
Saccharomyces cerevisiae
The C. alpina CREP1 was expressed in S. cerevisiae (w303–
1 A strain) with the plasmid p VT-CREP1 containing the
constitutive alcohol dehydrogenase promoter [3]. The D6
acetylenase (CER1)fromC. purpureus [12] was cloned
behind the galactose-inducible promoter GAL1 of the yeast
expression vector pYES2 (Invitrogen) generating the plas-
mids pYCER1 [12] and transformed in S. cerevisiae INVSc1
cells (Invitrogen) using the poly(ethylene glycol) method
[13].
Cell growth
Transformed yeast cells were grown in complete minimal
dropout-uracil medium (CMdum), i.e. YNB complete
medium (Sigma, St Louis, MO, USA) and complete
supplement mixture without uracil (Bio101,inc) with 2%
(v/v) glucose, a t 30 °C for 48 h. The cells were then
diluted to A
600
¼ 0.2 with fresh CMdum (total volume
10 mL). Fatty acids dissolved in 10% (v/v) Tween 40
(BDH Chemicals Ltd, Poole, UK) were then added to
the cultures transformed with pYCER1 to a final
concentration of 0.03% (w/v) fatty acids and 1% (w/v)
Tween 40. These cultures were incubated further for 8 h
after which expression of the CER1 gene was induced by
adding galactose to a final concentration of 1.8% (w/v).
The addition offatty acids in Tween 40 (at the same
concentrations as described above) was carried out after
8 h of incubation to th e cultures t ransformed with
CREP1. All cell cultures were then grown for additional
72 h before harvesting. The cells were subsequently
broken by a glass-bead shaker and their lipids extracted
into chloroform according to the method devised b y
Bligh and Dyer [14].
Fatty acid and lipid analysis
Polar lipids were sep arated from neutral lipids by thin l ayer
chromatography in hexane/diethyl ether/acetic acid
(70 : 30 : 1, v/v/v) on precoated silica gel 60 plates (Merck).
Gels from polar lipid areas (application spots) were scraped
off and methylated in situ with 0.1
M
sodium methoxide .
Fatty acid methyl esters of total lipids from chloroform
extracts of yeast cells were prepared by alkaline transme-
thylation using 2 mL of 0.1
M
sodium methoxide for 5 min
at 90 °C. Preparation of methyl esters from the seeds were
performed by treatment of whole seeds with 2 mL of 0.1
M
sodium methoxide for 55 min at 90 °C. Fattyacid methyl
esters were extracted with hexane and quantified by
GC using a WCOT fused silica capillary column
(50 m · 0.32 mm; film thickness, 0.25 lm), CP-wax 58
(FFAP)-CB (Chrompack, Middelburg, the Netherlands).
GC was performed on a Shimadzu GC-17 A equipped with
an autoinjector AOC-20i a nd an FID ( flame ionization
detector) detector using helium (4.0 mLÆmin
)1
)ascarrier
gas. The injector and detector temperatures were at 230 and
270 °C, respectively. Oven temperature was held at 165 °C
for 0.5 min and then raised to 25 0 °Cat4.0°CÆmin
)1
.
Heptadecanoic acid methyl ester was used as an internal
standard.
Fatty acid preparations
9(Z),12(E)-Octadecadienoic a cid w as prepared from cis -
12,13-epoxy-9(Z)-octadecenoic acid by hydrolysis into the
threo-12,13-diol f ollowed by syn elimination of the diol
using the Corey–Winter procedure [15]. The 9(Z),12(E)-
octadecadienoate was obtained in 41% yield and in > 98%
purity following preparative RP-HPLC. cis-12,13-Epoxy-
9(Z)-octadecenoic acid, 9(E),12(Z)-octadecadienoic acid
and 9(E),12(E)-octadecadienoic acid were purchased from
Larodan Fine Chemicals, Malmo
¨
, Sweden. [12(S)-
2
H]Oleic
acid was prepared from 12(R)-hydroxy-9(Z)-octadecenoic
acid (ricinoleic acid) using previously described m ethodo-
logy [16] (Fig. 1). The isotope composition was 96.2%
monodeuterated and 3.8% undeuterated molecules.
[(+/–)-erythro-12,13–
2
H
2
]Oleic acid was synthesized
from the methyl ester of cis-9,10-epoxy-12(Z)-octadecenoic
acid by catalytic deuteration using Wilkinson’s catalyst
followed by elimination of the cis-epoxide function using
triphenylphosphine selenide (Fig. 1). The material obtained
was purified by RP-HPLC to afford [(+/–)-erythro-
12,13-
2
H
2
]oleic acid in 55% yield. The isotope composition
was 97.0% dideuterated, 1 .4% monodeuterated and 1.6%
undeuterated molecules. It is well established that deuter-
ations using Wilkinson’s catalyst take place by clean syn
Fig. 1. Reaction used to prepare deuterated substrates. Reactionsusedtoprepare(A)[12(S)-
2
H]oleic acid and (B) [ erythro-12,13–
2
H
2
]oleic acid.
2992 A. S. Carlsson et al. (Eur. J. Biochem. 271) Ó FEBS 2004
addition of deuterium [17], i.e. deuteration of a Z double
bond will afford the erythro dideuterated product. Also the
triphenylphosphine selenide-promoted elimination takes
place stereospecifically, i.e. producing a Z alkene from a
cis-epoxide [18].
GC-MS and GLC analysis
GC-MS was carried out with a Hewlett-Packard model
5970B mass selective detector connected to a Hewlett-
Packard model 5890 gas chromatograph equipped with a
capillary column of Supelcowax (30 m ; film thickne ss
0.25 lm; carrier gas helium). Injections were made in the
split mode using an initial column temperature of 150 °C.
The temperature was raised at 3 °CÆmin
)1
until 250 °C.
GLC with flame ionization detection was carried out with
a Hewlett-Packard model 5890 gas chromatograph under
the same conditions as those used in GC-M S.
Identification of 9(
Z
),12(
E
)-octadecadienoic acid
An unknown 9,12-octadecadienoate produced in th e pres-
ence of recombinant C. alpina D12 acetylenase in A. thaliana
and yeast and also present in seeds of C. alpina was isolated
as its methyl ester by RP-HPLC using a solvent syst em of
acetonitrile/water 75 : 25 (v/v). The ester cochromato-
graphed w ith methyl 9(Z),12(E)- and 9 (E),12(Z)-octadeca-
dienoates (effluent volume, 65 mL) but separated from
methyl linoleate (59 mL) and methyl 9(E),12(E)-octadeca-
dienoate (71 mL). The material obtained was analyzed by
GLC. Its retention time was identical to that of methyl
9(Z),12(E)-octadecadienoate (20 min 30 s), but differed
from those of m ethyl 9(E),12(Z)-octadecadienoate (20 min
50s),methyl9(Z),12(Z)-octadecadienoate (20 m in 16 s),
and from those of a number of isomeric 9,11- and
10,12-octadecadienoates (22 min 58 s to 24 min 16 s).
Analysis by GC-MS was in full agreement with the
identification of the unknown octadecadienoate as methyl
9(Z),12(E)-octadecadienoate.
Results
The fattyacid composition of lipids from C. alpina and
C. palaestina seeds were compared with that of seeds from
wild type and the fad2 mutant as well as seeds from
A. thaliana lines transformed w ith either the C. alpina D12
acetylenase gene (CREP1)ortheC. palaestina epoxygenase
gene (CPAL2)(Table1).C. alpina seed lipids contained
significant amount (3.3%) of 9(Z),12(E)-octadecadienoate
(Materials and methods section), which have also been
reported earlier in Crepis rubra [19], whereas the seed lipids
from C. palaestina, wild type and the fad2 mutant did not
contain detectable amounts of this un usual stereoisomer of
18:2. In order to investigate if the synthesis of this E isomer
of 18:2 in C. alpina seed was a product of t he C. alpina D12
acetylenase activity, the acetylenase gene was expressed in
both wild type and t he fad2 mutant. Both transgenic lines
produced this fattyacid and the concentration was about
50% higher in the background of the fad2 mutant than in
the wild type background (3.5% vs. 2.3%) indicating that
oleate was the substrate for the formation of the D12E
double bond. It should be noted that the level of
18:2D9Z,12E was nearly four times higher than the level
of the acetylenic acid 18:1D9Z,12a (crepenynate) in t he wild
type transformed with CREP1.Inthefad2 mutant trans-
formed with CREP1, only trace amount of crepenynate was
detected. There was a significant increase of 18:2D9Z,12Z
in the fad2 line transformed with CREP1 compared with the
nontransformed mutant, indicating that the acetylenase also
could carry out D12(Z)desaturationofoleate.
We also analyzed the acyl composition of seed lipids
from cwild type and fad2 mutant transformed with the
C. palaestina D12 epoxygenase gene (CPAL2), which is
evolutionary closely related to the C. alpina acetylenase [3].
Small amounts of 18:2D9Z,12E were detected in both
transgenic lines (0.2%), and this was both 30 and nine times
less than the epoxy fatty acids formed by the epoxygenase in
the t ransformed wild type and fad 2 lines, r espectively
(Table 1). The wild type transformed with CPAL2 showed
Table 1. Acyl c omposition o f seeds from vild species and transgenic plant lines. Acyl composition in seeds from C. alpina, C. palaestina and wild type
and fad2 mutants of nontransforme d A. thaliana and transformed with either the C. alpina D12 acetylenase gene (CREP1)ortheC. palaestina
epoxygenase gene (CPAL2). nd, Not detected.
Fatty acids
Acyl composititon (area percentage)
C. alpina A. thaliana fad2
A. thaliana
+ CREPI
fad2
+ CREPI C. palaestina
A. thaliana
+ CPAL2
fad2
+ CPAL2
16:0 4.6 7.6 4.9 8.7 6.1 4.4 9.0 5.0
16:1 D9Z 0.1 0.3 0.3 0.3 0.4 0.1 0.4 0.5
18:0 2.0 4.6 2.6 3.2 2.7 2.5 4.3 3.0
18:1 D9Z 1.1 17.6 42.8 29.0 46.3 9.4 38.8 55.0
18:1 D11Z 0.9 1.2 1.7 2.3 2.2 0.5 1.8 3.0
18:2 D9Z,12Z 9.8 24.7 4.3 18.7 6.6 23.7 4.7 2.0
18:2 D9Z,12E 3.3 nd nd 2.3 3.5 nd 0.2 0.2
18:2 D9Z,15Z nd nd 0.7 nd 0.3 nd nd 0.4
18:3 D9Z,12Z,15Z nd 18.9 11.2 10.6 7.6 nd 6.7 4.0
20:1 D11Z nd 20.9 21.8 18.2 20.8 nd 21.8 18.0
18:1 D9Z, 12a 77.8 nd nd 0.6 Trace nd nd nd
12-Epoxy-18:1 D9Z nd nd nd nd nd 54.1 3.6 0.9
12-Epoxy-18:2 D9Z,15Z nd nd nd nd nd nd 2.1 0.9
Others 0.4 4.1 9.7 6.8 3.6 5.4 6.6 7.1
Ó FEBS 2004 Stereochemistry ofacetylenase/desaturaseenzymes (Eur. J. Biochem. 271) 2993
a drastic reduction in 18:2D9Z,12Z content compared with
the untransformed plants (4.7% vs. 24.7%) and in the fad2
line transformed with CPAL2, the 18:2D9Z,12Z content
was decreased from 4 % to 2%. It has p reviously been
shown that epoxy fattyacid production in A. thaliana
severely inhibits formation of linoleate and that the degree
of inhibition is correlated with the levels of the epoxy fatty
acids accumulated [11].
In order to further investigate t he catalytic activities of
the C. alpina acetylenase, we transformed baker’s yeast
(S . cerev isiae)withtheCREP1 gene. In contrast to
A. thaliana, S. cerev isiae lacks D12 desaturase activities
and can readily incorporate exogenous added fatty acids
into their lipids. In absence of exogenous fatty acids, the
yeast expressing the C. alpina acetylenase gene converted up
to 0.65% of their fatty acids into 1 8:2 in a ratio of D12(E)
to (Z) isomers of approximately 3 : 1 (Table 2). Similar
conversion rates were seen with yeast cultures fed o n
exogenously added oleate (Table 2). Only trace amounts
(0.01–0.02%) of 18:1D9Z,12a were produced in these
cultures. When linoleate was added t o the cultures, the
formation of the E isomer of 18:2 was still evident, although
its c oncentration was reduced by 80% (to 0.09%) and
0.53% of crepenynic acid was formed. When 18:2D9Z,12E
was added to the yeast transformed with CREP1,no
crepenynic acid was formed. As acyl groups linked to
phospholipids are the s ubstrate for the D12 desaturase-like
enzymes, we measured the acyl composition of the polar
lipids in the yeast after incubation with the exogenous fatty
acids. The l evels of t he two e xogenously s upplied 18:2
isomers were approximately the same in polar lip ids as in
total lipids, accounting for 49 and 41% of the total acyl
groups in the incubation with added (Z)and(E) isomer,
respectively (data not shown).
As it has previously been shown that a D6 acetylenase
(CER1) from the moss C. purpureus also has D6desatu-
rase activity [12], w e i nvestigated if that enzyme was
capable of also carry out E desaturation. Yeast trans-
formed with the CER1 was grown in presence of either
18:2D9Z,12Z or 18:2D9Z,12E (Table 3). The CER1
desaturated endogenous 16:1D9Z,18:1D9Z and exogenous
18:2D9Z,12Z to the corresponding D6( Z) derivatives
and also efficiently further converted the formed
18:3D6Z,9Z,12Z (c-18:3) to 18:3D6a,9Z,12Z. Exogenous
18:2D9Z,12E wasconvertedto18:3D6Z,9Z,12E but not
further to t he acetylenic compound. No D6(E) isomers
were found in the yeast transformed with the D6acetyl-
enase gene.
It was of particular interest to investigate the stereochem-
istry in the removal of hydrogen atoms in the formation of
the two geometrical isomers of 18:2 produced by CREP1.
Extracts containing fattyacid m ethyl esters obtained
following feeding of [12(S)-
2
H]- and [erythro-12,13-
2
H
2
]oleic
acids to yeast expressing the CREP1 gene were subjected to
GC-MS, and the isotope contents of methyl 9(Z),12(Z)- and
9(Z),12(E)-octadecadienoates were determined by selected
ion monitoring. Additionally, the isotope contents of oleates
recovered in the two experiments were determined. The
oleate recovered a fter feeding o f [12(S)-
2
H]oleate was a
mixture o f 39.7 deuterated and 60.3% undeuterated mole-
cules showing that the a dded oleate was diluted approxi-
mately 1.5-fold with unlabeled material during the
incubation period. A similar dilution was noted for the
oleate recovered after feeding of the [erythro-12,13-
2
H
2
]
oleate, i.e. 34.8% dideuterated, 0.6% monodeuterated, and
64.6% undeuterated molecules. As seen in Table 4,
9(Z),12(Z)- and 9(Z),12(E)-octadecadienoates both retained
most of the deuterium label w hen formed f rom [12(S)-
2
H]oleate, and accordingly, the 12(R) hydrogen was lost
from C-12 when the 12(Z)and12(E) double bonds were
introduced. F eeding of [erythro-12,13-
2
H
2
]oleates produced
9(Z),12(Z)-octadecadienoic acid, which was mainly either
undeuterated or dideuterated, had an isotopic composition
close to that expected for removal of the 12(R),13(R)
or 12(S),13(S) deuteriums (Table 5). In contrast, the
9(Z),12(E)-octadecadienoate mainly consisted of either
undeuterated or monodeuterated molecules in accordance
with a removal of the 12(R),13(S)or12(S),13(R)deute-
riums. By combining the results in Tables 4 and 5 it could be
deduced that the 9(Z),12(Z)-octadecadienoate was formed
by elimination of the pro-R hydrogen atoms at C-12 and
C-13 whereas the 9(Z),12(E)-octadecadienoate was formed
by elimination of the pro-R hydrogen at C-12 and the pro-S
hydrogen at C-13.
Table 2. Acyl composition in nontransformed (empty plasmid) yeast (S. cerevisiae ) and in yeast expressing D12 acetylenase gene (CREP1). Yeast was
grown in the absence and presence of e xogenous fatty acids. nd, Not detected.
Acyl group
Acyl composition (area percentage)
Empty plasmid
Fatty acid added
CREP1
Fatty acid added
None 18:1D9z 18:2D9Z,12Z 18:2D9Z,12E None 18:1D9z 18:2D9Z,12Z 18:2D9Z,12E
16:0 22.3 23.1 23.1 23.0 20.4 23.4 25.4 24.1
16:1D9Z 37.2 29.2 8.1 18.3 25.9 21.1 5.3 10.6
18:0 10.8 9.4 10.4 9.2 14.9 12.6 13.2 13.1
18:1D9Z 28.9 37.3 6.3 12.6 37.2 41.8 5.7 10.7
18:1D11Z 0.7 0.9 0.01 0.2 0.9 0.06 0.08 0.1
18:2D9Z,12Z nd nd 51.9 nd 0.18 0.19 49.3 nd
18:2D9Z,12E nd nd nd 36.6 0.47 0.41 0.09 41.0
18:1D9Z,12a nd nd nd nd 0.02 0.01 0.53 nd
Others 0.1 0.1 0.1 0.1 0.03 0.4 0.4 0.4
2994 A. S. Carlsson et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
We demonstrate that the C. alpina D12 acetylenase is a tri-
functional enzyme that efficiently produces a mixture of
D12 (Z)and(E) isomers of 18:2 from o leate as well as
18:1D9Z,12a (crepenynate) from l inoleate. The evolutionarily
closely related D12 epoxygenase from C. palaestina [11] was,
on the o ther hand, primarily an 18:2 epoxygenase utilizing
linoleate as the substrate, although it had also a weak
D12 (E) oleate desaturase activity when ex pressed in
A. thaliana. The evolutionarily distantly r elated bifunctional
D6 d esaturase/acetylenase from C. purpureus did not exhibit
(E) desaturase a ctivity, demonstrating that such activity is
not an inherent property of acetylenases.
The tra ns desaturation of oleate to 18:2D9Z,12E has
now been shown for three different D12 desaturase-like
enzymes, the C. alpina acetylenase a s shown here, the
conjugase from tung [7] and the desaturase from
Dimorphoteca sinuata [9]. In D. sinuata seeds, the D12(E)
desaturase produces the substrate for a second D12
desaturase-like enzyme converting 18:2D9Z,12E into
9-hydroxy-18:2D10E,12E, the dominating acyl group in
D. sinuata seeds. However, in C. alpina seeds the
18:2D9Z,12E is an end product, as we show here that it
cannot be converted further into crepenynic acid. Of all
the fattyacid desaturases so far reported it is only t he
C. alpina acetylenase that produces a m ixture of E and Z
isomers. However, a plant sphingolipid D8 desaturase that
converts 4-hydroxy-sphinganine into a mixture of 7 : 1 of
E and Z isomers o f 4-hydroxy-8-sphingenine has been
cloned and characterized [20]. A single fattyacid desatu-
rase enzyme has been implicated in both E and Z D11
desaturation of myristoyl-CoA in insects [21]. It is of
special interest that the two isomeric products of this
desaturation in insects are precursors for two different
pheromones with different biological activities. However,
no gene encoding a D11 desaturase yielding both E and Z
isomers has yet been cloned. The physiological significance
of the formation of 18:2D9Z,12E by C. alpina acetylenase
is unknown. The e nzyme serves to produce acetylenic fatty
acids, wh ich are most probably i nvolved in pathogen
Table 3. Acyl composition of nontransformed yeast (S. cerevisiae) and yeast expressing D6 acetylenase (CER1). Yeastweregrowninthepresenceof
18:2D9Z,12Z or 18:2:2D9Z,12E. nd, Not detected.
Fatty acids
Acyl composition (area percentage)
Empty plasmid
Fatty acid added
CER1
Fatty acid added
18:2D9Z,12Z 18:2D9Z,12E 18:2D9Z,12Z 18:2D9Z,12E
16:0 32.8 30.4 28.5 20.3
16:1D9Z 5.5 17.7 5.5 22.6
16:2D6Z,9Z nd nd 0.15 0.64
16:2D6E,9Z nd nd nd nd
18:0 10.0 10.9 11.5 12.2
18:1D9Z 7.7 21.4 8.1 30.1
18:1D11Z 0.16 0.61 0.12 0.90
18:2D6Z,9Z nd nd 0.05 0.22
18:2D6E,9Z nd nd nd nd
18:2D9Z,12Z 43.5 nd 44.8 nd
18:2D9Z,12E nd 18.9 nd 12.3
18:3D6Z,9Z,12Z nd nd 0.69 nd
18:3D6E,9Z,12Z nd nd nd nd
18:3D6Z,9Z,12Z nd nd nd 0.69
18:2D6a,9Z,12Z nd nd 0.57 nd
Others 0.34 0.09 0.02 0.05
Table 4. Isotope compositions of 9(Z),12(Z)- and 9(Z),12(E)-octa-
decadienoates generated from [12(S)-
2
H]oleic acid. The oleate given to
the cells had the following isotopic composition: 96.2% monodeuter-
ated, 3.8% undeuterated molecules, an d the oleate recovered consisted
of 39.7% deuterium-labeled and 60.3% unlabelled molecules due to
dilution with endogenous material.
d
0
(%) d
1
(%)
9(Z),12(Z)-octadecadienoate 36.4 63.6
9(Z),12(E)-octadecadienoate 34.4 65.6
Expected for R elimination 39.7 60.3
Expected for S elimination 0 100
Table 5. Isotope compositions of 9(Z),12(Z)- and 9(Z),12(E)-octa-
decadienoates generated from [erythro-12,13-
2
H
2
]oleic acid. The oleate
given to the cells had the following isotopic composition: 97.0%
dideuterated, 1.4% monodeuterated, 1.6% undeuterated molecules,
and the oleate recovered consisted of 34.8% dideuterated, 0.6%
monodeuterated, and 64.6% undeuterated molecules due to dilution
with endogenous material.
d
0
(%) d
1
(%) d
2
(%)
9(Z),12(Z)-octadecadienoic acid 18.5 0.3 81.2
9(Z),12(E)-octadecadienoic acid 2.0 30.6 67.4
Expected for R,R or S,S elimination 17.4 0.3 82.3
Expected for R,S or S,R elimination 0 35.1 64.9
Ó FEBS 2004 Stereochemistry ofacetylenase/desaturaseenzymes (Eur. J. Biochem. 271) 2995
defense mechanisms [4], however, other functions of the
enzyme are conceivable.
Stereochemical and mechanistic studies have been per-
formed on the trans and cis desaturation by the myristoyl-
CoA desaturase [21], the sphingolipid desaturase [20] and
of the formation of the acetylenic bond by the C. alpina
acetylenase [22]. Our studies using stereospecifically deuter-
ated oleates revealed that the desaturations leading to the
12(Z) a nd 12(E) alkenes by the C. alpina acetylenase
proceeded with distinct ste reochemistries. Thus, the Z
double bond-forming reaction resulting in 9(Z),12(Z)-
octadecadienoic acid took place by selective removal o f
the pro-R hydrogen atoms from C-12 and C-13, a result that
was in accord with previous studies of other Z-desaturases
[21, 23, 24]. Formation of the E double bond of 9(Z),12(E)-
octadecadienoic acid took place by selective removal o f
the pro-R hydrogen from C-12 and the pro-S hydrogen
from C-13.
It is well established from studies of kinetic isotope effects
[20–24] that en zymatic desaturations take place in a stepwise
manner and involve an initial, slow hydrogen abstraction
followed by rapid collapse of the intermediate thus formed
and expulsion of the second hydrogen atom. Importantly,
because of the short lifetime of the singly desaturated
intermediate, it is unlikely that a conformational change can
take place p rior to eliminatio n of t he second hyd rogen
atom. It seems v ery likely that this mechanism is valid also
for the Crepis alpina D
12
acetylenase/desaturase studied in
the presen work. Although the three-dimensional structure
of this enzyme is unknown, it is possible t o rationalize the
stereochemical results in terms of the different conforma-
tions the oleate substrate has to adopt t o generate e ither a
12(Z)ora12(E) double bond. Thus, it can be postulated
that the C
11
–C
14
segment of the oleate carbon chain has to
be in the cisoid conformation to produce the 9(Z),12(Z)-
octadecadienoate and in the transoid conformation to
produce the 9(Z),12(E)-octadecadienoate (Fig. 2). In the
biosynthesis of the 9(Z),12(Z) isomer, the pro-R hydrogens
at C
12
and C
13
will be in close contact w ith the hydrogen-
abstracting groups on the enzyme surface and be eliminated.
In order to produce the 9(Z),12(E) isomer, the C
12
–C
13
single bond has to rotate to produce the transoid confor-
mation of the C
11
–C
14
segment. As a consequence of this
rotation, the pro-R hydrogen at C-13 will move away from
the hydrogen-abstracting group whereas the pro-S hydrogen
will come in contact and be eliminated (Fig. 2).
A comparison between the end products produced by the
D6 acetylenase and the D12 acetylenase in y east expressing
genes for these enzymes revealed an interesting difference.
When linoleate was fe d to the y east expressing the D6
acetylenase, the products of the desaturation reactions, i.e.
18:3D6Z,9Z,12Z and 18:2D6a,9Z,12Z accounted for about
1.3% of all fatty acids in the cell and of which 40% was
found as the acetylenic fattyacid and thus had undergone
two rounds of desaturation catalyzed by the acetylenase.
Although this efficient conversion of 18:3D6Z,9Z,12 Z to
18:2D6a,9Z,12Z. Thus the product of the first desaturation
round was efficiently competing with the about 40 times
higher concentration of linoleate in the membranes.
Although a D6(Z) desaturase gene from C. purpureus has
been cloned [12], it i s t hus plausible that much of the
18:3D6Z,9Z,12Z used for the production of the acetylenic
fatty acid is generated by the acetylenase enzyme itself in this
moss. T his would be consistent with the very low levels of
18:3D6Z,9Z,12Z compared with linoleate found in phos-
phatidylcholine (the site for D6 desaturation and acetylena-
tion) in C. purpureus [25].
The C. alpina acetylenase, on the other hand, does not
have such a metabolic channeling. Of the s mall amount of
linoleate produced by the acetylenase in yeast, only 5–10%
is converted further to 18:1D9Z,12a. It is interesting to note
that 18:2D9Z,12E, which cannot be converted further into
18:1D9Z,12a, amounts to 75% of the products of oleate
desaturation catalyzed by the C. alpina acetylenase. The
C. alpina seed lipids (mainly triacylglycerols) contain about
3% of 18:2D9Z,12E and nearly 80% of 18:1D9Z,12a. Thus,
the acetylenase is responsib le for the production of about
1% of 18:2D9Z,12Z in C. alpina seedsandtherestofthe
linoleate produced in these seeds, which is 86% of all acyl
groups in the seed (% linoleate +% crepenynate –%
linoleate formed by the acetylenase), must be a result of
D12(Z) oleate desaturase activity catalyzed by a separate
enzyme. Because the C. alpina acetylenase appears as
efficient as a desaturase as an acetylenase, this n early total
out competing of the acetylenase by the D12(Z)desaturase
for oleate substrate in C. alpina seeds appears at fi rst sight
puzzling. However, results presented here using cells having
a different ratio of oleate to linoleate indicate that this ratio
determines the ratio of utilization of the two substrates by
the acetylenase. The ratio of o leate to linoleate in C. alpina
seeds is 1 : 10 and under such conditions a d esaturation to
acetylenation ratio of about 1 : 20 would be expected based
on ou r results in transgenic organisms. This is close to the
ratio that could be calculated on the basis of the amount of
18:2D9Z,12E and 18:1D6a,9Z found in these seeds. The
conjugase from tung also has, in addition to its conjugase
activity with linoleate, a high oleate D12(E) desaturase
activity [7]. The tung seed lipids has about 3%
of 18:2D9Z,12E and 80% of a-eleostearic acid (18:3D9Z,
11E,13E) and it is probable that the relative proportion of
oleate and linoleate in these seeds also determines the
relative rate of utilization of these two substrates by the
conjugase [7]. However it is not known if 18:2D9Z,12E is an
end product or if it could be used by the conjugase for
the synthesis of a-eleostearic acid.
Fig. 2. Conformations of oleic ac id bound to C. alpina D12 acetylenase.
The conformation in (A) will prod uce 9(Z),12(Z)-octadecadienoic acid
and the one in (B) will produce 9(Z),12( E)-o ctadecadienoic acid.
2996 A. S. Carlsson et al. (Eur. J. Biochem. 271) Ó FEBS 2004
The data presented here and by Sperling et al. [12] show
that the D6 acetylenase can utilize a variety of D9
desaturated a cyl groups as substrates for the first desatu-
ration reaction, such as 16:1D9Z, 18:1D9Z, 18:2 D9Z,12Z,
18:2D9Z,12Eand18:3D9Z,12Z,15Z. However, the forma-
tion of acetylenic bonds is only occurring with substrates
having a D12(Z) double bond. Thus in order f or the double
bond to be orientated properly t owards the active site f or
further hydrogen subtraction, a D12 double bond in (Z)
configuration seems essential.
The r esult p resented in this paper is an additional
demonstration of the great flexibility in substrate acceptance
and catalytic outcome of the fattyacid desaturase enzymes.
In case of the D6 acetylenase from the moss C. purpureus,
this implies that the enzyme not only produces D6acetylenic
fatty acids in this species, but also provides its own D6
desaturated substrate f or this reaction. In contrast, the
C. alpina D12 acetylenase produces mainly a dead-end
substrate from oleate and thus must obtain its substrate for
the a cetylenase r eaction of a separate D12 des aturase
enzyme.
Acknowledgement
Financial su pport from the Swedish University of Agricultural Sciences
strategic research grant ÔThe Biologica l F actory/Agr iFunGenÕ,Swedish
Oil Growers Association (SSO), VL-stiftelsen, and the Swedish
Research Council for Environment, Agricultural Sciences and Spatial
Planning (project number 2001–2553) are gratefully acknowledged.
Arabidopsis fad2 seeds transformed with CREP1 and CPAL1 genes
were kindly provided by Drs Allan Green and Surinder Singh at
CSIRO, Canberra, Australia.
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Ó FEBS 2004 Stereochemistry ofacetylenase/desaturaseenzymes (Eur. J. Biochem. 271) 2997
. Properties of two multifunctional plant fatty acid
acetylenase/desaturase enzymes
Anders S. Carlsson
1
, Stefan Thomaeus
1
,. for about
1.3% of all fatty acids in the cell and of which 40% was
found as the acetylenic fatty acid and thus had undergone
two rounds of desaturation