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 transformed
Trang 1Properties of two multifunctional plant fatty acid
acetylenase/desaturase enzymes
Anders S Carlsson1, Stefan Thomaeus1, Mats Hamberg2and Sten Stymne1
1
Department of Crop Science, Swedish University of Agricultural Sciences, Alnarp, Sweden;2Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden
The properties of the D6 desaturase/acetylenase from the
moss Ceratodon purpureus and the D12 acetylenase from the
dicot Crepis alpina were studied by expressing the encoding
genes in Arabidopsis thaliana and Saccharomyces cerevisiae
The acetylenase from C alpina D12 desaturated both oleate
and linoleate with about equal efficiency The desaturation
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-Rhydrogen atoms were removed from C-12 and C-13
in the introduction of the 12(Z) double bond, whereas the
pro-Rand pro-S hydrogen atoms were removed from these
carbons during the formation of the 12(E) double bond The
results suggested that the D12 acetylenase could
accommo-date oleate having either a cisoid or transoid conformation
of the C12-C13single bond, and that these conformers served
as precursors of the 12(Z) and 12(E) double bonds,
respectively However, only the 9(Z),12(Z)-octadecadieno-ate isomer could be further desatur9(Z),12(Z)-octadecadieno-ated 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 acyl groups possessing a D9 double bond as substrates The D6 double bond was efficiently further converted 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 of plant 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 and stereochemistry of 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) and 12(Z) isomers of
18:2 The catalytic activity of the C alpina acetylenase
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 thaliana 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)2Æs)1,
20C 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
inA 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 of pBI121 under the transcriptional control 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 fatty acid analyses of the transformants were performed on T 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)
Trang 2Expression of D6- and D12-desaturase-related enzymes
inSaccharomyces cerevisiae
The C alpina CREP1 was expressed in S cerevisiae (w303–
1 A strain) with the plasmid pVT-CREP1 containing the
constitutive alcohol dehydrogenase promoter [3] The D6
acetylenase (CER1) from C 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, at 30C for 48 h The cells were then
diluted to A600¼ 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 of fatty acids in Tween 40 (at the same
concentrations as described above) was carried out after
8 h of incubation to the cultures transformed 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 by
Bligh and Dyer [14]
Fatty acid and lipid analysis
Polar lipids were separated from neutral lipids by thin layer
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 0.1M 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 0.1Msodium methoxide for 5 min
at 90C Preparation of methyl esters from the seeds were performed by treatment of whole seeds with 2 mL of 0.1M sodium methoxide for 55 min at 90C Fatty acid 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 and an FID (flame ionization detector) detector using helium (4.0 mLÆmin)1) as carrier gas The injector and detector temperatures were at 230 and
270C, respectively Oven temperature was held at 165 C for 0.5 min and then raised to 250C at 4.0 CÆmin)1 Heptadecanoic acid methyl ester was used as an internal standard
Fatty acid preparations 9(Z),12(E)-Octadecadienoic acid was prepared from cis-12,13-epoxy-9(Z)-octadecenoic acid by hydrolysis into the threo-12,13-diol followed 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)-2H]Oleic acid was prepared from 12(R)-hydroxy-9(Z)-octadecenoic acid (ricinoleic acid) using previously described methodo-logy [16] (Fig 1) The isotope composition was 96.2% monodeuterated and 3.8% undeuterated molecules [(+/–)-erythro-12,13–2H2]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-12,13-2H2]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 Reactions used to prepare (A) [12(S)-2H]oleic acid and (B) [erythro-12,13–2H ]oleic acid.
Trang 3addition 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 thickness
0.25 lm; carrier gas helium) Injections were made in the
split mode using an initial column temperature of 150C
The temperature was raised at 3CÆmin)1 until 250C
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-MS
Identification of 9(Z),12(E)-octadecadienoic acid
An unknown 9,12-octadecadienoate produced in the
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 system of
acetonitrile/water 75 : 25 (v/v) The ester
cochromato-graphed with 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 methyl 9(E),12(Z)-octadecadienoate (20 min
50 s), methyl 9(Z),12(Z)-octadecadienoate (20 min 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 fatty acid composition of lipids from C alpina and
C palaestinaseeds were compared with that of seeds from wild type and the fad2 mutant as well as seeds from
A thalianalines transformed with either the C alpina D12 acetylenase gene (CREP1) or the C palaestina epoxygenase gene (CPAL2) (Table 1) 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 unusual 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 the C alpina D12 acetylenase activity, the acetylenase gene was expressed in both wild type and the fad2 mutant Both transgenic lines produced this fatty acid 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 the wild type transformed with CREP1 In the fad2 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) desaturation of oleate
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 transformed wild type and fad2 lines, respectively (Table 1) The wild type transformed with CPAL2 showed Table 1 Acyl composition of seeds from vild species and transgenic plant lines Acyl composition in seeds from C alpina, C palaestina and wild type and fad2 mutants of nontransformed A thaliana and transformed with either the C alpina D12 acetylenase gene (CREP1) or the C 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
Trang 4a 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 previously been
shown that epoxy fatty acid 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 the catalytic activities of
the C alpina acetylenase, we transformed baker’s yeast
(S cerevisiae) with the CREP1 gene In contrast to
A thaliana, S cerevisiae 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 18:2 in a ratio of D12(E)
to (Z) isomers of approximately 3 : 1 (Table 2) Similar
conversion rates were seen with yeast cultures fed on
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 to the cultures, the
formation of the E isomer of 18:2 was still evident, although
its concentration 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 substrate 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 levels of the two exogenously supplied 18:2
isomers were approximately the same in polar lipids 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 D6
desatu-rase activity [12], we investigated 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 was converted to 18:3D6Z,9Z,12E but not further to the acetylenic compound No D6(E) isomers were found in the yeast transformed with the D6 acetyl-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 fatty acid methyl esters obtained following feeding of [12(S)-2H]- and [erythro-12,13-2H2]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 after feeding of [12(S)-2H]oleate was a mixture of 39.7 deuterated and 60.3% undeuterated mole-cules showing that the added 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-12,13-2H2] 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 when formed from
[12(S)-2H]oleate, and accordingly, the 12(R) hydrogen was lost from C-12 when the 12(Z) and 12(E) double bonds were introduced Feeding of [erythro-12,13-2H2]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) or 12(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 exogenous 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
Trang 5We 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 oleate as well as
18:1D9Z,12a (crepenynate) from linoleate The evolutionarily
closely related D12 epoxygenase from C palaestina [11] was,
on the other hand, primarily an 18:2 epoxygenase utilizing linoleate as the substrate, although it had also a weak D12(E) oleate desaturase activity when expressed in
A thaliana The evolutionarily distantly related bifunctional D6 desaturase/acetylenase from C purpureus did not exhibit (E) desaturase activity, demonstrating that such activity is not an inherent property of acetylenases
The trans desaturation of oleate to 18:2D9Z,12E has now been shown for three different D12 desaturase-like enzymes, the C alpina acetylenase as 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 fatty acid desaturases so far reported it is only the
C alpinaacetylenase that produces a mixture 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 of 4-hydroxy-8-sphingenine has been cloned and characterized [20] A single fatty acid 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 enzyme serves to produce acetylenic fatty acids, which are most probably involved in pathogen
Table 3 Acyl composition of nontransformed yeast (S cerevisiae) and yeast expressing D6 acetylenase (CER1) Yeast were grown in the presence of 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
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, and the oleate recovered consisted
of 39.7% deuterium-labeled and 60.3% unlabelled molecules due to
dilution with endogenous material.
d 0 (%) d 1 (%)
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
Trang 6defense 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) and 12(E) alkenes by the C alpina acetylenase
proceeded with distinct stereochemistries Thus, the Z
double bond-forming reaction resulting in
9(Z),12(Z)-octadecadienoic acid took place by selective removal of
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 of
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 enzymatic 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 prior to elimination of the second hydrogen
atom It seems very likely that this mechanism is valid also
for the Crepis alpina D12acetylenase/desaturase studied in
the presen work Although the three-dimensional structure
of this enzyme is unknown, it is possible to rationalize the
stereochemical results in terms of the different
conforma-tions the oleate substrate has to adopt to generate either a
12(Z) or a 12(E) double bond Thus, it can be postulated
that the C11–C14segment 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 C12and C13will be in close contact with the
hydrogen-abstracting groups on the enzyme surface and be eliminated
In order to produce the 9(Z),12(E) isomer, the C12–C13
single bond has to rotate to produce the transoid
confor-mation of the C11–C14 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 yeast expressing genes for these enzymes revealed an interesting difference When linoleate was fed to the yeast 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 fatty acid and thus had undergone two rounds of desaturation catalyzed by the acetylenase Although this efficient conversion of 18:3D6Z,9Z,12Z 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 is thus 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 This 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 small 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 alpinaseed lipids (mainly triacylglycerols) contain about 3% of 18:2D9Z,12E and nearly 80% of 18:1D9Z,12a Thus, the acetylenase is responsible for the production of about 1% of 18:2D9Z,12Z in C alpina seeds and the rest of the 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 nearly total out competing of the acetylenase by the D12(Z) desaturase for oleate substrate in C alpina seeds appears at first 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 oleate to linoleate in C alpina seeds is 1 : 10 and under such conditions a desaturation to acetylenation ratio of about 1 : 20 would be expected based
on our 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 acid bound to C alpina D12 acetylenase.
The conformation in (A) will produce 9(Z),12(Z)-octadecadienoic acid
and the one in (B) will produce 9(Z),12(E)-octadecadienoic acid.
Trang 7The data presented here and by Sperling et al [12] show
that the D6 acetylenase can utilize a variety of D9
desaturated acyl groups as substrates for the first
desatu-ration reaction, such as 16:1D9Z, 18:1D9Z, 18:2D9Z,12Z,
18:2D9Z,12E and 18:3D9Z,12Z,15Z However, the
forma-tion of acetylenic bonds is only occurring with substrates
having a D12(Z) double bond Thus in order for the double
bond to be orientated properly towards the active site for
further hydrogen subtraction, a D12 double bond in (Z)
configuration seems essential
The result presented in this paper is an additional
demonstration of the great flexibility in substrate acceptance
and catalytic outcome of the fatty acid desaturase enzymes
In case of the D6 acetylenase from the moss C purpureus,
this implies that the enzyme not only produces D6 acetylenic
fatty acids in this species, but also provides its own D6
desaturated substrate for 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 acetylenase reaction of a separate D12 desaturase
enzyme
Acknowledgement
Financial support from the Swedish University of Agricultural Sciences
strategic research grant The Biological Factory/AgriFunGen, 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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