Revealing the catalytic potential of an acyl-ACP
desaturase: Tandem selective oxidation of
saturated fatty acids
Edward J. Whittle*, Amy E. Tremblay†, Peter H. Buist,†and John Shanklin*‡
*Department of Biology, Brookhaven National Laboratory, 50 Bell Avenue, Upton, NY 11973; and†Department of Chemistry, Carleton University,
1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6
Edited by Wendell Roelofs, Cornell University, Geneva, NY, and approved July 14, 2008 (received for review June 10, 2008)
It is estimated that plants contain thousands of fatty acid struc-
tures, many of which arise by the action of membrane-bound
desaturases and desaturase-like enzymes. The details of ‘‘unusual’’
e.g., hydroxyl or conjugated, fatty acid formation remain elusive,
because these enzymes await structural characterization. How-
ever, soluble plant acyl-ACP (acyl carrier protein) desaturases have
been studied in far greater detail but typically only catalyze
desaturation (dehydrogenation) reactions. We describe a mutant
of the castor acyl-ACP desaturase (T117R/G188L/D280K) that
converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-
18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of
regiospecifically deuterated substrates shows that the conversion
of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via
hydrogen abstraction at C-11 and highly regioselective hydroxy-
lation (>97%) at C-9.18O-labeling studies show that the hydroxyl
oxygen in the reaction product is exclusively derived from molec-
ular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two
major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid
isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be
rationalized by differences in substrate binding as dictated by the
curvature of substrate channel at the active site. That three amino
acid substitutions, remote from the diiron active site, expand the
the membrane-bound desaturase family underscores the latent
potential of O2-dependent nonheme diiron enzymes to mediate a
diversity of functionalization chemistry. In summary, this study
contributes detailed mechanistic insights into factors that govern
the highly selective production of unusual fatty acids.
binuclear iron ? diiron ? hydroxylation ? nonheme iron ? catalysis
fication reactions are initiated by hydrogen removal from an
unactivated methylene/vinyl group and result in oxidation of
substrate (2). In all reactions described to date, an activated
oxygen species formed at a diiron center (3, 4) is believed to
effect the initial hydrogen abstraction. The recognition of this
coworkers (5) to identify the gene encoding the oleoyl 12-
hydroxylase based on its homology to the oleoyl 12-desaturase.
Appreciation of the catalytic diversity of desaturase-like en-
zymes (6) stimulated a search for, and subsequent identification
of, an acetylenase, epoxygenase (7), and enzymes responsible for
the synthesis of conjugated fatty acids (8) and allylic alcohols (9).
Progress toward understanding the determinants of reaction
outcome has been achieved via comparative studies on the
oleate-desaturase/hydroxylase pair involving systematic site-
directed mutagenesis of residues proximal to the putative active
site (10, 11). Unfortunately, further interpretation of these data
will not be possible until this class of membrane-bound enzymes
is structurally characterized.
Several crystal structures (12–15) are available for the struc-
turally distinct, soluble acyl-acyl carrier protein (ACP) desatu-
t has been estimated that the seeds of plants contain thousands
of structurally distinct fatty acids (1). Many fatty acid modi-
rases (6, 16–18) but this class of enzyme acts only as a desaturase
with natural substrates and precludes the desired correlation of
structure with a range of reaction outcomes. The acyl-ACP
family of desaturases contains a number of members with
different substrate chain length specificities and regioselectivi-
ties (6, 19). However, the absence of reported oxygenation
striking. In the present work, we describe a triple mutant of the
castor desaturase that is able to convert stearoyl- or oleoyl-ACP
to the allylic alcohol (E)-10-18:1-9-OH with high regioselectiv-
ity; the (E)-isomer of oleoyl-ACP, elaidyl-ACP, is converted to
a mixture of (Z)-10-18:1-9-OH and the conjugated 9-(E)-11-(Z)
isomer of linoleic acid (CLA). The different reaction outcomes
are rationalized by distinct substrate binding modes in a curved
Results and Discussion
Mutant Desaturase Converts Stearoyl Substrate to a Hydroxylated
Product. During the course of experiments designed to under-
stand the factors governing regioselectivity, we engineered a
triple mutant T117R/G188L/D280K of the castor ?9 desaturase
(Fig. 1) (referred to in this article as ‘‘mutant desaturase’’) in an
attempt to mimic structural features of the ivy ?4 desaturase
(14). The expected change in regioselectivity from ?9 to ?4 was
not observed; however, the 18:0 substrate was converted into
(Z)-9-18:1 product, which initially accumulated and then disap-
peared, as indicated by GC analysis (Fig. 2 A–C). The apparent
mass balance deficit was restored upon derivatization of the
product mixture with (N,O-bis[Trimethylsily]trifluoroac-
etamide) plus trimethylchlorosilane, whereupon a ?9:1 mixture
of two new products (Fig. 2D) was observed. This result sug-
gested that these novel products were alcohols. The mass spectra
of the trimethylsilyl (TMS) derivatives of the major and minor
hydroxylated product (Fig. 2 E and F) were indistinguishable
from each other and featured an apparent molecular ion (m/z ?
384) consistent with a C18 product containing an O-TMS group
and a double bond. The presence of an m/z 227 ion in these mass
spectra is diagnostic for an O-TMS group at C-9 and a double
bond between C-9 and the methyl group of the fatty acid. The
initial formation of (Z)-9-18:1-ACP, and its subsequent decrease
in abundance with hydroxy product accumulation indicated that
(Z)-9-18:1-ACP was a substrate for the mutant enzyme. This
interpretation was confirmed by the observation of hydroxylated
Author contributions: J.S. designed research; E.J.W., A.E.T., and J.S. performed research;
A.E.T. and P.H.B. contributed new reagents/analytic tools; E.J.W., A.E.T., P.H.B., and J.S.
analyzed data; and P.H.B. and J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 23, 2008 ?
vol. 105 ?
product formation upon incubating (Z)-9-18:1-ACP with the
mutant desaturase; the ratio of the two hydroxy components
mimicked that obtained previously with 18:0-ACP substrate
H-Abstraction from C-11 of Oleoyl Substrate Leads to the Highly
Regioselective Formation of the 10-18:1-9OH Allylic Alcohol. The
position of hydrogen abstraction in oleyl ACP oxidation was
identified by incubating dideuterated acyl-ACPs with the mutant
desaturase and mass spectral analysis of the hydroxyl products
(Table 1). The 9,10-d2-oleyl- (Cambridge Isotope Labs) and
11d2-oleyl-ACPs (20) were prepared from available labeled oleic
acid precursors, but 12d2-oleyl-ACP was generated in situ from
12-d2-stearoyl-ACP (20). Gas chromatography-mass spectrom-
etry analysis of the products obtained from these incubations
revealed that one deuterium was removed when 11-d2-oleoyl-
ACP was presented as substrate, whereas both deuterium atoms
were retained in the product when the label was present at C-9,
C-10, or C-12. Consideration of these results, together with the
mass spectral data discussed previously, led us to propose an
allylic alcohol structure, namely (Z)- or (E)-10-18:1-9-OH for
the novel hydroxyl products obtained from this mutant desatu-
rase. To confirm the position of the hydroxyl group and to assign
the stereochemistry of the double bond in this product mixture,
we synthesized four standards [(Z)- and (E)-10-18:1-9-OH, (Z)-
and (E)-9-18:1-11-OH) using standard synthetic routes [see the
supporting information (SI)]. A comparison of the mass spectra
is shown in yellow.
Location of residues T117, G188, and D280 in the WT desaturase. The
monoenes. (A–D) Gas chromatographic separation of FAMES obtained from
reaction mixtures at reaction time t ? 0 (A), t ? 0.5 h (B), and t ? 5 h (C and
D). C and D display chromatograms obtained before and after TMS derivati-
zation, respectively. Fatty acid methyl esters: 1, 18:0; 2, (Z)-9-18:1; 3, minor
product peak; 4, major product peak, *contaminant. (E and F) Mass spectra
correspond to the peaks labeled 3 and 4, respectively.
Mutant desaturase converts 18:0-ACP into a pair of novel hydroxy
isomers of the 10-18:1-9-OH allylic alcohol. Gas chromatographic separation
of methyl esters following TMS derivatization displays the conversion of
(Z)-9-18:1-ACP substrate to product (A), synthetic (Z)-10-18:1-9-OH standard
(B), synthetic (E)-10-18:1-9-OH standard (C), combination of samples A and B
(D), and combination of samples A and C (E). Numbers on the gas chromato-
grams have the same designation as in Fig. 2. (F and G) Mass spectra corre-
the synthetic standard (E)-10-18:1-9-OH in C, respectively, are shown.
Mutant desaturase product peaks are identified as (Z)- and (E)-
Table 1. Mass Spectral data for 10-18:1-9-OH, TMS derivative
obtained from incubation of various deuterated substrates with
Substrate Molecular ion
Major* fragment ion of
*Fragment represents ?95% of the total ion current.
†Molecular ion of product (10-18:1-9-OTMS) methyl ester.
‡Mass of fragment ion due to cleavage between carbons 8 and 9.
Whittle et al.
September 23, 2008 ?
vol. 105 ?
no. 38 ?
of the TMS derivatives of these standards with those of the
reaction products reveal an excellent match for (Z)- or (E)-10-
spectrum of (Z)- and (E)-9-18:1-11-OH features an entirely
different fragmentation pattern attributable to cleavage ? to the
O-TMS group between C-11 and C-12 (data not shown). This
analysis leaves (Z)- and (E)-10-18:1-9-OH as the only possible
candidates for the enzymatically generated allylic alcohols.
Elucidation of the stereochemistry of these compounds was
accomplished by a comparison of their GC elution times with
those of our reference standards (Fig. 3 B and C). Spiking of
the product mixture (Fig. 2D) with the (Z)-10-18:1-9-OH
standard resulted in enhancement of the faster eluting peak
labeled 3; the product peak spiked with the (E)-isomer is
shown in Fig. 3E. Thus, the major (?90%) product is
(E)-10-18:1-9-OH, and the minor (?10%) product is the
Based on these results and the consensus mechanism for the
WT enzyme (2) (Fig. 4A), we postulate that a hydrogen atom is
abstracted at C-11 of oleyl-ACP by the oxidant; the resultant
allylic radical is trapped regioselectively at C-9 by the iron-
hydroxyl species (Fig. 4B). The curvature of the active site
hinders rotation around the C-10, C-11 bond and yields primarily
Elaidyl Substrate Is Converted to a Mixture of an Allylic Alcohol and
a Diene. Our discovery that (Z)-9-18:1-ACP can act as a substrate
for a soluble ?9 desaturase variant is without precedent and
offered a unique opportunity to probe the structural boundaries
of a well characterized active site with stereochemically defined
olefinic substrates. We asked whether (E)-9-18-ACP could act as
a substrate with the mutant enzyme. Indeed, although a poorer
substrate than the (Z) isomer (Table 2), (E)-9-18:1-ACP was
converted primarily to a mixture of allylic alcohols and a diene
component. The former consisted primarily of (Z)-10-18:1-
9-OH along with minor amounts of (E)-10-18:1-9-OH (?10%)
and coeluting (E)-9-18:1-11-OH (?3%) (determined by GC-MS
spiking and single ion monitoring experiments using reference
standards as described previously; data not shown). In addition
to the allylic alcohols, a novel product produced in approxi-
mately equimolar ratio featured a molecular ion of m/z 294,
suggesting that it is a dienoic C-18 fatty acid (Fig. 5C). Based on
analysis of the mass fragmentation ladder of its pyrrolidine
derivative (Fig. 5D) (21), the positions of the double bonds in the
dienoic product were determined to be C-9 and C-11 (i.e., the
product is a CLA).
To determine the stereochemistry of the double bonds, we
compared the GC elution time of the product with those of 18:2
synthetic standards [(E)-9,(Z)-11/(Z)-9,(Z)-11 and (Z)-9,(E)-11/
(E)-9,(E)-11], all of which had different retention times (Fig. 6
B and D). Spiking of the enzymic product with these standards
allowed us to identify the dienoic product as the (E)-9,(Z)-11
isomer (Fig. 6C)—a product of (Z)-selective desaturation at
The Hydroxyl Oxygen Is Derived from O2. The mechanistic pathway
by which oleoyl-ACP is converted to the (E)-isomer of 9-OH
18:1?10 by the mutant acyl-ACP desaturase is reminiscent of
that proposed for the biosynthesis of dimorphecolic acid from
oleic acid (9)—a sequence that is thought to involve oxygenation
of an allylic radical intermediate. Performing desaturase incu-
bations in the presence of18O2allowed us to establish the origin
(Z)-9-18:1 (A) and (E)-10-18:1-9-OH (B) formation are shown in the context of the bend in the desaturase substrate binding channel (gray) adjacent to the active
site oxidant. (C) Postulated substrate binding modes of the nonnatural elaidyl substrate that leads to the formation of (Z)-10-18:1-9-OH and (E)-9-(Z)-11-18:2 are
Scheme illustrates the proposed substrate binding modes and reaction outcomes for various substrates with mutant desaturase. The mechanisms of
Table 2. Kinetic parameters of the castor desaturase mutant
T117R/G188L/D280K and wt enzyme
KM, ? M
*kcatis reported per diiron site.
†Mutant is T117R/G188L/D280K.
‡Mean with standard error in parentheses.
www.pnas.org?cgi?doi?10.1073?pnas.0805645105Whittle et al.
of oxygen atom in the allylic alcohol products. This approach is
potentially capable of distinguishing between direct molecular
oxygen-derived hydroxyl rebound to a radical intermediate or
quenching of a carbocation by an active site water molecule (22).
Thus, enzyme incubations were performed under an
atmosphere for both (Z)- and (E)-9-18:1 substrates. The
respective (E)- and (Z)-9-OH 18:1 product isomers were
analyzed by MS (Table 3), and it was found that ?98% of
the oxygen in the hydroxyl groups in both products originated
from molecular oxygen. The exclusive (within experimental
error) incorporation of O2-derived oxygen into the (E)- and
(Z)-10-18:1-9-OH products is consistent with a mechanism
involving hydroxyl rebound to an allylic radical (Fig. 4B). We
recognize, however, that capture of an allylic carbocation by
active site water derived from molecular oxygen remains a
Reaction Outcome. As described previously, the T117R/G188L/
D280K mutant castor desaturase, like the WT enzyme, converts
18:0-ACP to (Z)-9-18:1-ACP. However, in contrast to the WT
desaturase, the (Z)-9-18:1 moiety is further converted to mainly
(E)-10-18:1-9-OH by the mutant. Notably, the 9-(E)-18:1 sub-
strate is primarily converted to the 10-(Z)-9-OH product. We
have yet to cocrystallize the substrates in the mutant enzyme
successfully and are therefore not able to correlate substrate
complexes with the observed products; however, the available
desaturase crystal structures provide a useful context within
which to rationalize the observed product stereochemistries. A
with a bend adjacent to the active site as predicted by Bloch (23)
and revealed in subsequent crystal structures (12, 13). In the WT
desaturase, the shape of the cavity (Fig. 1) forces the saturated
with pro(R) hydrogens facing the diiron center (24). The avail-
able evidence (24–26) suggests that the site of initial oxidation
may be at C-10 for WT enzyme (Fig. 4A). The T117R/G188L/
D280K mutant enzyme allows the (Z)-9-18:1 substrate to bind in
the active site because of changes remote from the diiron active
site (Fig. 1) that might affect positioning of the pantetheine
group linking the ACP to the fatty acid substrate (residue 280)
or the methyl terminus of the fatty acyl portion of the substrate
(residues 117 and 188). A possible binding mode for (Z)-9-18:1
is shown in Fig. 4B (compare with the binding of the natural
18:0-substrate shown in Fig. 4A). We envision hydrogen abstrac-
CLA. Gas chromatographic separation of methyl esters following TMS deriva-
tization of reaction mixtures containing (E)-9-18:1 substrate either before (A)
or after (B) the addition of mutant desaturase enzyme. Fatty acid methyl
9-(E),11-(Z)-18:2 CLA. (C) Mass spectrum of methyl ester peak 5 is shown. (D)
Mass spectrum of the pyrrolidine derivative of peak 5 is shown.
enzyme CLA product. (B) Standard mix of 1, (E)-9-(Z)-11-18:2, and 2, (Z)-9-(Z)-11-
18:2. Asterisk indicates residual eneyne starting material (see the SI). (C) Combi-
4, (E)-9, (E)-11-18:2. (E) Combination of methyl esters from A and D.
(A) Gas chromatographic separation of methyl esters of: the mutant
Whittle et al.
September 23, 2008 ?
vol. 105 ?
no. 38 ?
tion to proceed at the nearest available methylene group, namely
C-11. The hydroxyl rebound occurs exclusively (?99.5%) at C-9
of the allylic radical along with the formation of a double bond
between C-10 and C-11 that is principally in the (E)-
configuration because of the curvature of the active site in this
region. Thus, for this soluble desaturase mutant, like the
previously reported case of membrane dimorphecolic acid-
forming enzyme (9), hydrogen abstraction occurs at C-11 and
the hydroxyl group is added at C-9. That the allylic 9-OH is the
sole product implies that positioning of the (Z)-9-18:1 sub-
strate must be precise; if it were not, we would perhaps expect
to observe the formation of some 11-OH-9-ene product.
Interestingly, 9-epoxy-stearate or the acetylenic compound,
stearolic acid (9-octadecynoic acid) [i.e., products of oxidation
observed in related enzymes (6)] are not detected (?0.5%), as
judged by spiking experiments with authentic standards.
Further support for the hypothesis that the curvature of the
active site adjacent to the diiron center is a primary determinant
of reaction outcome was obtained from the incubation of
(E)-9-18:1 with the mutant desaturase. According to our model,
incomplete insertion of the acyl chain would be expected (Fig.
4C), such that collapse of the allylic radical intermediate would
yield primarily a 9-hydroxylated product bearing a 10-(Z) double
bond as is observed. With this substrate, a minor amount of
11-hydroxylated (?3%) product is also formed by hydroxyl
rebound to the site of initial hydrogen abstraction, and this
compound bears a 9E double bond as anticipated. The concom-
itant formation of the conjugated diene suggests two possible
binding modes of the (E)-9-18:1 substrate (Fig. 4C). The sub-
strate complex favoring the introduction of the conjugated
double bond is envisaged to arise from insertion of the fatty acyl
chain slightly less deeply than that favoring allylic alcohol
formation. In this mode, C-11 and C-12 are at the bend in the
binding cavity, which dictates a quasi-eclipsed substrate confor-
mation similar to that postulated for C-9 and C-10 of stearate in
the WT enzyme, leading to the same configurational outcome
[i.e., the (Z)-double bond] (Fig. 4 A and C). Precedence for this
sort of regiochemical ‘‘error’’ product has been obtained with
unnatural substrates (24, 27). The two binding-mode hypothesis
and the stereochemistry of H-abstraction and hydroxyl rebound
are the subject of current investigation.
The WT desaturase converts 18:0-substrate into 18:1 product,
with no further oxidation. We have preliminary data that
18:1-ACP can inhibit the rate of desaturation, suggesting that it
is capable of binding to the WT desaturase, but the absence of
product formation suggests that the necessary conformational
changes associated with redox gating are not triggered (28). How
the three amino acid changes allow the binding and subsequent
oxidation of 18:1-ACP in the mutant enzyme awaits the results
of structural studies. Although the mutant castor desaturase
described is a member of the soluble desaturase family, the
sequential oxidation of stearoyl-ACP described herein is some-
what similar to the three consecutive oxidations of 16:0-CoA by
a processionary moth pheromone gland membrane-bound
The present study demonstrates the catalytic plasticity of diiron
chemistry for a mutant soluble desaturase in which only three
amino acids, remote from the active site, differ from that found
in the WT sequence. In the mutant, the natural substrate
18:0-ACP is converted in a two-step oxidation reaction to mainly
(E)-10-18:1-9-OH via a (Z)-9-18:1 intermediate. Incubation of a
nonnatural substrate, (E)-9-18:1-ACP, resulted in the produc-
tion of (Z)-10-18:1-9-OH and an (E)-9, (Z)-11 CLA isomer.
Formation of these products can be rationalized in terms of
distinct binding modes for the substrates that are conformation-
ally constrained by curvature of the active site. That a triple
mutant of the soluble desaturase expands the range of reaction
outcome mimics that seen for the integral membrane class of
desaturases and underscores the latent potential of O2-
dependent nonheme diiron enzymes to mediate a rich variety of
functionalization chemistry. The data presented here provide
novel and detailed mechanistic information on the factors
governing the highly selective production of three unusual fatty
Materials and Methods
Mutant Construction. A triple mutant T117R/G188L/D280K was engineered
from the WT castor desaturase with the use of overlap extension PCR (30) to
produce the D280K mutation with the use of the following pairs of primers,
D280Kf gataatctttttaaacacttttcagctgttgcgc and D280Kr gcgcaacagct-
gaaaagtgtttaaaaagattatc, in conjunction with flanking pET9d primers. The
fragment was introduced into the pET9d backbone after restriction with
XbaI and EcoRI and cloning into the equivalent unique sites of pET9d. The
T117R and G188L mutations were introduced by restriction of desaturase
clone 5.2 (31) with NcoI and Acc65I and introduction of this fragment into
the equivalent sites in the D280K mutant desaturase containing pET9d
clone. Introduction of the T117R/G188L/D280K mutations was confirmed
Fatty Acid Analysis. Desaturase enzyme was generated by expression from a
pET9d construct in Escherichia coli BL21(DE3) cells, followed by enrichment to
?90% purity by 20CM cation exchange chromatography (Applied Biosys-
tems). Fatty acid desaturase reactions (600 ?l) were performed by incubation
of the desaturase with 18:0-ACP and 18:1-ACP substrates in the presence of
recombinant spinach ACP-I as previously described (32). Several deuterium-
Isotope Laboratories) or from the collection of Tulloch (20). Enzyme reactions
were terminated by the addition of 1 ml of toluene. Fatty acid methyl esters
incubation for 60 min at 50°C. The FAMES were extracted twice into 2 ml of
was evaporated to dryness under a stream of N2, and resuspended in ?100 ?l
of hexane in preparation for GC analysis. For analysis of hydroxy fatty acids,
the FAMEs were evaporated to dryness and resuspended in 100 ?l of (N,O-
bis[Trimethylsily]trifluoroacetamide plus trimethylchlorosilane (Supelco) for
45 min at 60°C to convert the hydroxy fatty acids to their trimethyl silyl
derivatives. The FAMEs and their TMS derivatives were analyzed with the use
with 60-m ? 250-?m SP-2340 capillary columns (Supelco). The oven temper-
ature was raised from 100°C to 160°C at a rate of 25°C min?1and from 160°C
to 240°C at a rate of 10°C min?1with a flow rate of 1.1 ml/min?1. Mass
spectrometry was performed with an HP5973 mass selective detector
(Hewlett-Packard). Authentic standards were prepared as described in the SI.
For the18O experiments, oxygen was removed from the sample cell by
repeated purging of the sample cell with O2-free argon and vacuum with the
use of a Schlenk line. Two samples were prepared, one containing desaturase
phate (NADPH)-positive reductase, and substrate, and the other containing
ferredoxin and NADPH. Both samples were degassed by the application of 15
cycles of vacuum/argon; at that time;18O2(98%) (Cambridge Isotope Labora-
tories) was introduced into the sample cell containing the desaturase and the
degassed ferredoxin/NADPH was introduced to initiate the reaction. The
Table 3. Incorporation of18O into the hydroxyl group of the
(Z)-10-18:1-9-O TMS(E)-10-18:1-9-O TMS
Each incubation was carried out three times with ?85% conversion; per-
deviations are indicated in parentheses. Isotopic content was calculated by
integration of mass spectral peaks at m/e 227/229, corrected for natural
abundance isotopes. nd, not determined.
www.pnas.org?cgi?doi?10.1073?pnas.0805645105 Whittle et al.
reaction was terminated by the introduction of toluene esterified and sily-
lated as described previously.
ACKNOWLEDGMENTS. E.J.W. and J.S. thank the Office of Basic Energy Sci-
ences of the U.S. Department of Energy, for financial support of this work.
Support was also provided by Natural Sciences and Engineering Research
Council (NSERC) (CGS-M postgraduate scholarship program to A.E.T., Grant
OGP-2528 to P.H.B.). We thank Pat Covello for providing some of the deuter-
ated compounds and Jodie Guy for structural comparisons of ivy and castor
desaturase and for graphics assistance.
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