Báo cáo Y học: Mechanism of 1,4-dehydrogenation catalyzed by a fatty acid (1,4)-desaturase of Calendula officinalis pptx

Mechanism of 1,4-dehydrogenation catalyzed by a fatty acid
(1,4)-desaturase of
Calendula officinalis
Darwin W. Reed
1
, Christopher K. Savile
2
, Xiao Qiu
1
, Peter H. Buist
2
and Patrick S. Covello
1
1
Plant Biotechnology Institute, Saskatoon, SK, Canada;
2
Department of Chemistry, Carleton University, Ottawa, Ontario, Canada
The mechanism by which the fatty acid (1,4)-desaturase of
Calendula officinalis produces calendic acid from linoleic acid
has been probed through the use of kinetic isotope effect
(KIE) measurements. This was accomplished by incubating
appropriate mixtures of linoleate and regiospecifically
dideuterated isotopomers with a strain of Saccharomyces
cerevisiae expressing a functional (1,4)-desaturase. GC-MS
analysis of methyl calendate obtained in these experiments
showed that the oxidation of linoleate occurs in two discrete
steps since the cleavage of the C11-H bond is very sensitive to
isotopic substitution (k
H
/k
D

¼ 5.7 ± 1.0) while no isotope
effect (k
H
/k
D
¼ 1.0 ± 0.1) was observed for the C8-H bond
breaking step. These data indicate that calendic acid is pro-
duced via initial H-atom abstraction at C11 of a linoleoyl
substrate and supports the hypothesis that this transforma-
tion represents a regiochemical variation of the more com-
mon C12-initiated D
12
desaturation process.
Keywords: desaturase; kinetic isotope effect; conjugated
fatty acid; deuterium labelling; Calendula.
The D
12
-oleate desaturase (FAD2) family of enzymes are
membrane-bound nonheme iron-containing proteins that
carry out a fascinating array of related oxidative transfor-
mations [1,2]. The prototypical reaction features the intro-
duction of a cis-double bond at the 12,13-position of an
oleoyl substrate – a ubiquitous biosynthetic reaction of
higher plants [3,4] (Fig. 1A). Species-specific mechanistic
variations of this process include 12-hydroxylation of oleate
(Ricinus communis) [5] and 12,13-epoxidation (Crepis
palaestina) [2] or 12,13-acetylenation (Crepis alpina)[2]of
linoleate. Recently, it has been shown that the production of
conjugated trienoic acids, such as calendic acid from a
linoleate precursor are also carried out by FAD2 variants

[6–8](Fig. 1B). The latter reaction is particularly noteworthy
given the current interest in conjugated fatty acids with
respect to their role in human nutrition [9] as well as
commercial applications [10].
As part of ongoing research into the structure–function
relationships of FAD2 type enzymes, a closer examination
of calendate formation is clearly warranted. Early labelling
experiments using marigold seed homogenates and labelled
linoleate precursors demonstrated that calendic acid is
produced by an apparent (1,4)-dehydrogenation process
whereby a linoleoyl substrate loses one hydrogen from C8
and C11, respectively [11]. No oxygenated intermediates
were detected. These results as well as related substrate
specificity data [6] point to a mechanism which is analogous
to that proposed for the more common (1,2)-dehydrogen-
ation reactions of fatty acid desaturases (Fig. 2). The
mechanistic model [12] for the latter process features an
initial, energetically difficult hydrogen abstraction step,
which generates a very short-lived, carbon-centered radical
intermediate, or its iron-bound equivalent (not shown). This
species collapses rapidly to give an unsaturated product by
what is formally a second hydrogen abstraction, although a
one electron oxidation/proton removal sequence cannot be
rigorously excluded at this time. The stepwise nature of this
transformation is supported by kinetic isotope effect (KIE)
studies of several membrane-bound fatty acid desaturases.
In all cases examined, one C-H cleavage was found to be
subject to a large primary deuterium kinetic isotope effect
while the second C-H bond rupture was insensitive to
isotopic substitution [12–22]. This pattern of KIEs is

precisely what one would expect for a disproportionation
mechanism [23] of the type showed in Fig. 2 and the data
were used to pinpoint the site of the initial oxidative attack
(ÔcryptoregiochemistryÕ) for these systems. In several cases,
additional independent evidence is available to support the
cryptoregiochemical assignments. That is, the location of
the putative diiron oxidant relative to substrate can be
ascertained by inducing the desaturase to behave as a
regioselective oxygenase through modifications of substrate
or enzyme [19,20,24–26].
The availability of a convenient yeast expression system
for Fac2 –aCalendula officinalis gene encoding the (1,4)-
desaturase involved in calendic acid production [6,8] offered
a unique opportunity to study the mechanism of this
reaction using KIE methodology. Specifically, we wished to
correlate the site of initial oxidation for this process with
that determined for D
12
-desaturation [13]. We report here,
the results of our collaborative investigation.
Correspondence to P. H. Buist, Department of Chemistry,
Carleton University, 1125 Colonel By Drive, Ottawa, Ontario,
Canada, K1S 5B6.
Fax: + 1 613 5203749 or 3830, Tel.: + 1 613 5202600 Ext. 3643,
E-mail:
or P. S. Covello, National Research Council, Plant Biotechnology
Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9.
Fax: + 1 306 9754839, Tel.: + 1 306 9755269;
E-mail:
Abbreviations: KIE, kinetic isotope effect.

Definition: The term (1,4)-desaturase denotes an enzyme that converts
an isolated carbon-carbon double bond in a fatty acid into two
conjugated double bonds by what is formally a 1,4-dehydrogenation
reaction. Such enzymes have also been termed conjugases [7].
(Received 23 July 2002, accepted 28 August 2002)
Eur. J. Biochem. 269, 5024–5029 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03209.x
EXPERIMENTAL PROCEDURES
Materials
Methyl linoleate (> 99%) was purchased from Nu-Chek-
Prep, Inc. The two regiospecifically dideuterated methyl
linoleates ([8,8
2
H
2
]-1,11,11-
2
H
2
]-1) required for the KIE
study were prepared by routes which were very similar to
those reported previously for the synthesis of the corres-
ponding chiral monodeutero analogues [27]. Thus, the
tosylate of 8-hydroxy-[8,8
2
H
2
]octanoic acid was reacted
with lithium acetylide-ethylenediamine complex to give
[8,8
2

H
2
]dec-9-ynoic acid which was in turn coupled with the
tosylate of 2-octyn-1-ol to give [8,8
2
H
2
]-1 after semihydro-
genation and methyl esterification of the intermediate diyne.
In a similar manner, dec-9-ynoic acid was C-alkylated with
the tosylate of [1,1-
2
H
2
]-2-octyn-1-ol to give [11,11-
2
H
2
]-1
after semihydrogenation and methyl esterification. The
overall yields of [8,8
2
H
2
]-1 and [11,11-
2
H
2
]-1 obtained via
these procedures was 5% and 14%, respectively. Purifica-

tion of substrates was carried out by flash chromatography
(silica gel, 0.5% v/v ethyl acetate/hexane) and HPLC
fractionation as previously described [16]. GC-MS analysis
[16] of the final deuterated products revealed that each
isotopomer consisted essentially entirely of dideuterated
species (m/z 296; 294 for nondeuterated analogue).
1
Hand
13
C NMR analysis confirmed the position of the two
deuterium atoms for each isotopomer as indicated by the
presence/absence of the diagnostic bisallylic signals at d
2.77 p.p.m. (
1
H) and 25.67 p.p.m. (
13
C) for [11,11-
2
H
2
]-1,
respectively, and an approximately two-fold attenuation of
the overlapping allylic signals (C-8,C-13) at d 2.02 p.p.m.
(
1
H) and 27.25 p.p.m. (
13
C) for [8,8
2
H

2
]-1.
Incubation experiments
For characterization of the Calendula fatty acid conjugase,
the yeast strain DTY10-a2 (MATa, fas2D::LEU2, can1-
100, ura3-1, ade2-1, his3-11, his3-15) [28] was transformed
with a plasmid (pYJ) comprised of the pYES2 vector
(Invitrogen) and the Fac2 gene from Calendula officinalis
[6] to give the strain pYJ/DTY10-a2. While not a
requirement for the experiments described, this strain
harbours a mutation in fas2, rendering it unable to
synthesize fatty acids de novo. Consequently, it was
routinely grown in minimal media containing galactose
[29],  0.1 m
M
pentadecanoic acid and 0.1% Tergitol type
NP-40 (a polymer of ethylene oxide and p-nonylphenol).
For fatty acid feeding experiments, cultures were supple-
mented with  0.3 m
M
substrate fatty acids and incubated
at 20 °C for three days followed by 15 °C for three days.
Yeast cultures (D
600
 4) were pelleted by centrifugation
(4000 g, 10 min) and pellets were washed with 10 mL 1%
Tergitol solution and 2 · 10 mL H
2
O prior to lipid
extraction.

Analytical procedures
For fatty acid analysis, yeast pellets were saponified by
adding 2 mL 10% KOH/methanol and heating at 80 °Cfor
2 h. The mixture was then cooled and pre-extracted with
2 · 2 mL hexane to remove nonsaponifiable lipids. The
reaction mixture was then neutralized with 50% acetic acid
to  pH 5 and the fatty acids were extracted with 2 · 2mL
hexane. The hexane was removed under a nitrogen stream
and the mixture, including the conjugated fatty acids, was
esterifiedwith2mL1%H
2
SO
4
in methanol at 50 °Cfor1
h (This methylation method has been found to be most
suitable for conjugated fatty acid ester analysis; W. Christie,
Mylnefield Research Services Ltd., Dundee, Scotland,
personal communication.) The cooled mixture was extrac-
tedwith2· 2 mL hexane. The pooled hexane was washed
with 2 mL H
2
O and concentrated under N
2
for HPLC
purification, GC or GC-MS analysis.
GC-MS analysis of yeast lipids was performed using a
Fisons VG TRIO 2000 mass spectrometer (VG Analytical,
UK) controlled by Masslynx version 2.0 software, coupled
to a GC 8000 Series gas chromatograph as previously
described [16] except that a narrow EI

+
scan range of 285–
305 m/z was used. A representative mass spectrum of
biosynthetic methyl calendate is shown in Fig. 3.
RESULTS AND DISCUSSION
Our methodology for determining the intermolecular
primary deuterium KIE on each C-H cleavage step in fatty
acid desaturation reactions involves GC-MS analysis of
olefinic products derived from direct competition between
nondeuterated substrate and the appropriate regiospecifi-
cally dideuterated (-C
2
H
2
-) fatty acid. As has been pointed
out on a previous occasion [13], the magnitude of the
primary deuterium KIE determined in this manner must be
regarded as an estimate since the observed value may
incorporate a small (< 10%) a-secondary isotope effect
[40]. In addition, partial masking of the ÔintrinsicÕ KIE by
Fig. 1. Biosynthesis of linoleate and calendate catalyzed by (1,2) and
(1,4)-desaturases, respectively. OPL, phospholipid ester; X, undefined
headgroup.
Fig. 2. Generic mechanistic scheme showing
the stepwise removal of hydrogens in fatty acid
(1,2)-desaturation. Structure of the putative
diiron oxidizing species is speculative.
Ó FEBS 2002 Calendic acid biosynthesis (Eur. J. Biochem. 269) 5025
other enzymic steps in the catalytic cycle such as substrate
binding may also be occurring [35]. None of these

considerations affect the conclusions reached in this paper.
The use of a competitive rather than a noncompetitive
experimental design has allowed KIE determinations to be
carried out for both in vitro and in vivo desaturase systems.
The results have correlated well with KIE data obtained by
other methods [30–34]. Our methodology dictates that
interference by endogenous d
0
-substrate, if present, must be
eliminated: this has been accomplished previously through
the use of unnatural chain-shortened substrates or ana-
logues bearing a remote ÔthiaÕ- or deuterium mass label.
Such measures proved unnecessary in the case of the
linoleate-calendate reaction since the host yeast system used
for this purpose does not biosynthesize the relevant
substrate.
Optimal incubation conditions for our KIE studies were
set up in a preliminary experiment: methyl linoleate 1
(100 mgÆL
)1
) was administered to cultures (50 mL) of the
pYJ/DTY10a2 strain of S. cerevisiae incubated at 20 °Cfor
3 days to permit relatively rapid growth and then at 15 °C
for a further 3 days to reach saturation at a temperature
which has been found to give better substrate conversion
rates. The cells were harvested by centrifugation and the
lipids were isolated via a hydrolysis/methylation sequence
known to be suitable for conjugated fatty acid esters (10%
w/v KOH/CH
3

OH, 80 °C, 2 h; neutralization to  pH 5
with acetic acid, hexane extraction, 1% w/v H
2
SO
4
/
CH
3
OH, 50 °C, 1 h). Analysis of the fatty acids as methyl
esters by GC-MS revealed that exogenous linoleate had
been converted to calendate to the extent of  1% of total
cellular fatty acids, a result similar to that observed
previously [6,8]. Control experiments previously indicated
that the production of 2 was dependent on expression of the
FAC2 enzyme.
The two regiospecifically dideuterated linoleates
([8,8-
2
H
2
]-1, [11,11-
2
H
2
]-1) required for the KIE study
(Fig. 4) were prepared via established synthetic routes (See
Experimental section). A mixture of each deuterated
material with its nondeuterated parent (1 mg) was admini-
stered to growing cultures (10 mL) of the S. cerevisiae
transformant (pYJ/DTY10-a2) using conditions identical to

that of the trial experiment. The deuterium content of the
olefinic fatty acid methyl esters in the cellular lipid extract
was assessed by GC-MS as described in the Experimental
section. The d
2
/d
0
ratio of the linoleate isotopomers found
in the cells was essentially identical to that of the starting
material in both incubations, as is required for these types of
competitive KIE measurements [35]. No loss of label due to
reversible exchange of deuterated linoleate at C-8 or C-11
could be detected. Mass spectral analysis of the calendate
fraction revealed that in both incubations, this material
consisted entirely of a d
0
/d
1
mixture indicating a loss of one
deuterium from the d
2
-substrate as expected. Product
kinetic isotope effects (k
H
/k
D
) were calculated using the
ratio: [% d
0
(product)/% d

1
(product)]/[% d
0
(substrate)/%
d
2
(substrate)] and this analysis indicated the presence of a
large primary deuterium isotope effect (5.7 ± 1.0, average
of four experiments) for the carbon-hydrogen bond clea-
vage at C11 while the C8-H bond breaking step was shown
to be essentially insensitive to deuterium substitution
(KIE ¼ 1.0 ± 0.1, average of four experiments) (Table 1).
According to our mechanism (Fig. 2), these results demon-
strate that calendate production is initiated by an energeti-
cally difficult and hence isotopically sensitive hydrogen
abstraction at C11 and completed by a second facile and
kinetically unimportant hydrogen abstraction at C8. The
fast formation of an allylic radical at C8 followed by rate-
determining hydrogen abstracton at C11 cannot be rigor-
ously excluded but seems far less likely given the intrinsically
high energy content of radical intermediates relative to
product.
Some decades ago, Morris and Marshall [36] speculated
that conjugated trienoic fatty acids are produced in plants
from linoleic acid via an allylic radical intermediate.
Crombie and coworkers [11] provided evidence that calen-
dic acid was indeed biosynthesized from linoleic acid via
removal of hydrogens at C8 and C11. More recently, it was
suggested that C8 might be the site of initial oxidation for
this process based on a comparison with the putative site of

initial attack catalyzed by a soluble plant D
9
desaturase [37].
However our results clearly demonstrate that calendate
production is in fact initiated at C11 as might be expected
for a process which is catalyzed by a homolog of FAD2 – an
enzyme which initiates the conversion of oleate to linoleate
at C12 [12]. Thus, the switch between 1,2 and 1,4-
dehydrogenation could conceivably be controlled by a
fairly small change in oxidant position relative to substrates
which both adopt a conformation allowing syn removal of
Fig. 4. Isotopomers of 1 used to probe the kinetic isotope effects on the
fatty acid (1,4)-desaturase reaction involved in calendate biosynthesis.
Fig. 3. Mass spectrum of biosynthetic methyl calendate. Arrow indi-
cates the molecular ion cluster used to calculate the isotopic content of
deuterated samples.
5026 D. W. Reed et al. (Eur. J. Biochem. 269) Ó FEBS 2002
two proximal hydrogens (H-H distance in both cases
 2.5 A
˚
). This model (Fig. 5) can be tested by determining
the stereochemistry of H-removal for calendate formation
using chiral monodeutero probes [27] and comparing this
result with the known pro-R enantioselectivity at C12,13
observed for D
12
-desaturation [38].
Further evidence for the close relationship between 1,2
and 1,4-dehydrogenation has been obtained recently for a
Spodoptera littoralis desaturating system which converts

11(Z)- tetradecenoate to 10(E),12(E)-tetradecadienoate by
initial H-abstraction at C10 and 11(E)-tetradecenoate
to 9(Z),11(E)-tetradecadienoate by initial oxidative attack
at C9 [39]. Whether these two transformations are
catalyzed by separate enzymes in this case remains to be
determined.
In summary, the cryptic site of initial oxidation for an
important plant fatty acid conjugase-mediated reaction has
been determined to be at the carbon furthest from C-1. In
contrast, all other desaturase-catalyzed oxidations studied
to date are initiated at the carbon closest to the acyl
headgroup. Thus it would be interesting to apply our KIE
methodology to the study of related FAD2-like enzymes [7]
involved in the formation of a-eleostearic acid
[9(Z),11(E),13(E)-octadecatrienoic acid] and a-parinaric
acid [9(Z),11(E),13(E),15(Z)-octadecatetraenoic acid] from
linoleic acid. In so doing, we would hope to correlate the
various regioselectivities observed for this important set of
catalysts with the geometric relationship between oxidant
and substrate.
Note added in proof: Recent site-directed mutagenesis
experiments using a D
12
desaturase/hydroxylase system have
validated the mechanistic paradigm underlying our crypto-
regiochemical determinations [41].
ACKNOWLEDGEMENTS
We wish to thank the National Science and Engineering Research
Council (NSERC) for financial support of the synthetic work
performed at Carleton University (PHB), Steve Ambrose for perform-

ing the GC-MS analysis, Charles Martin for providing the yeast strain
DTY-10a2 and Michele Loewen and Robert Sasata for reviewing the
manuscript.
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Fig. 5. Mechanistic model showing the rela-
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desat-
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Products
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c
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