Topological study of mechanistic diversity in conjugated fatty acid biosynthesis

Article abstract of DOI:10.1002/anie.201202080

Variations on an oxidative theme: The precision with which FAD2-type desaturases carry out C-H activation reactions on flexible lipidic substrates is astonishing. The conformational space available within the active site of these enzymes has been explored using deuterium-labeled substrates, and evidence for a novel quasi-eclipsed conformer has been uncovered. The scheme shows some prototypical substrate conformations. Copyright

Full text of DOI:10.1002/anie.201202080

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DOI: 10.1002/anie.201202080
Enzyme Mechanisms
Topological Study of Mechanistic Diversity in Conjugated Fatty Acid
Biosynthesis**
Palash Bhar, Darwin W. Reed, Patrick S. Covello,* and Peter H. Buist*
Plant-derived seed oils are of current interest in the context of
renewable hydrocarbon-type industrial feedstocks.[1,2] Func-
tionalization of oleochemicals adds value to this commodity
and is achieved by a set of O2-dependent, non-heme diiron
enzymes known as desaturases.[3–5] The FAD2 subfamily is
particularly interesting because of the mechanistic diversity
exhibited by this set of sequence-related membrane proteins
(Scheme 1).
acetylenation (pathway e),[10,11] and regioselective 1,4-dehy-
drogenation (pathway f,g).[12,13] The products of this phyto-
chemical virtuosity typically exhibit antifeedant bioactivity
and accumulate to concentrations as high as 90% in mature
plant seeds. The oxidative power and selectivity displayed by
FAD2-type enzymes is unmatched by any other known
catalytic system and deserves closer scrutiny.
The high sequence similarity of FAD2 variants suggests
that relatively modest changes in active site architecture are
sufficient to govern reaction outcome. The membrane-bound
nature of these enzymes has prevented their detailed
structural characterization but one can imagine that subtle
alterations to the contours of a hydrophobic binding pocket
could influence substrate positioning relative to a potent
oxidizing diiron(IV) species. To gain more information on this
aspect of FAD2 biochemistry, we decided to focus our
mechanistic study on three reactions that involve a common
11-H abstraction step. These are calendate (pathway f), a-
eleostearate (pathway g), and dimorphecolate formation
(pathway b). As FAD2 enzymes are highly unstable outside
their cellular membrane-bound state, we took advantage of
the fact that S. cerevisiae expression systems are available for
each enzyme[8,13,14] to carry out our mechanistic studies.
We began by studying the mechanistic relationship
between calendate and a-eleostearate biosynthesis
(Scheme 2). Using the KIE (kinetic isotope effect) method,
we have previously determined that the site of initial
H abstraction is at C11 in the 1,4-dehydrogenation process
leading to calendate.[15] We hypothesized that a-eleostearate
formation in the seeds of the tung tree (Aleurites fordii
Hemsl.) is also initiated at C11, but in this case, the putative
Scheme 1. Mechanistic diversity of the FAD2 family of desaturases.
The parent FAD2-catalyzed reaction involves initial pro-
R hydrogen atom abstraction at C12 of an oleyl substrate
ꢀ
followed by facile C13 HR-bond cleavage to form linoleate
(9Z,12Z-18:2).[6] The putative diiron oxidant is thought to be
structurally related to compound Q, the species believed to be
[7]
ꢀ
responsible for C H activation in methane monooxygenase.
ꢀ
This is a syn process and is consistent with both hydrogen
atoms being abstracted by the same iron-bound oxygen atom
as implied in Scheme 1. Species-specific variations of this
prototypical reaction include (12E)-olefin formation and
subsequent allylic oxidation[8] (pathway a,b), 12-hydroxyl-
ation (pathway c),[9] 12,13-epoxidation (pathway d),[10] 12,13-
radical intermediate would undergo rapid C14 H cleavage
rather than at C8 as shown in Scheme 2.
[*] P. Bhar, Prof. P. H. Buist
Department of Chemistry, Carleton University
1125 Colonel By Drive, Ottawa, ON K1S 5B6 (Canada)
E-mail: peter_buist@carleton.ca
D. W. Reed, Dr. P. S. Covello
NRC Plant Biotechnology Institute
110 Gymnasium Place, Saskatoon, SK S7N 0W9 (Canada)
E-mail: patrick.covello@nrc-cnrc.gc.ca
[**] We thank Dauenpen Meesapyodsuk, John Dyer, Tony Kinney, and Ed
Cahoon for providing plasmids. This project was funded by NSERC
Grant OGP-2528(PHB).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202080.
Scheme 2. Biosynthesis of calendate and a-eleostearate.
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The site of initial oxidation (cryptoregiochemistry) for a- eleostearate formation is initiated by hydrogen abstraction at
eleostearate formation was elucidated by measuring the C11 and completed by a second facile hydrogen abstraction at
primary deuterium kinetic isotope effects on each of the the C14 position (Scheme 2). It is significant that in both
ꢀ
two C H cleavage steps. This is a general approach that has calendate and a-eleostearate formation, the oxidant attacks
been validated and applied successfully to some sixteen 11-H of linoleate first. This implies that the different
desaturases.[5] The method is based on the premise that the regiochemical outcome (8,10-diene vs. 11,13-diene) formation
ꢀ
cleavage of the first C H bond in desaturase-mediated is simply dictated by oxidant access to C8 versus C14 of
reactions is energetically more difficult than the subsequent a common bisallylic radical intermediate in the second H-
ꢀ
collapse of the putative radical intermediate by b-C H abstraction event. This might be achieved by regioselective
scisson. In principle, only the first step should be rate-limiting cleavage of either iron–oxygen bond in the first hydrogen
and subject to a primary deuterium KIE. Typically KIE values abstraction step with concomitant movement of the iron(IV)
are determined by intermolecular competitive experiments hydroxy group towards either the C1 or C18 terminus of
using 1:1 mixtures of non-deuterated and regiospecifically substrate (Scheme 2).
dideuterated substrates.[16]
To further investigate the topology of 1,4-desaturation, we
mixture consisting of approximately equivalent wished to determine the stereochemistry of calendate and a-
A
amounts of saponified methyl esters of non-labeled and eleostearate formation. To this end, we prepared the six
[11,11-D2]linoleates or [14,14-D2]linoleates[17] was introduced, possible stereospecifically labeled 8-[D1], 11-[D1], and 14-
in triplicate, as a solution in 10% Tergitol (ca. 5 mg each) to [D1]linoleates and examined the fate of the deuterium label
growing cultures (50 mL) of the pDR100/INVSC1 strain of S. upon 1,4-desaturation of the appropriate set of compounds by
cerevisiae (see the Supporting Information), a transformant Calendula officinalis D8,10-desaturase and Aleurites fordii
that functionally expresses the D11,13-desaturase enzyme from Hemsl. D11,13-desaturase. The Crombie-based[19] synthetic
Aleurites fordii Hemsl.[13] The incubation conditions (208C for route to the required linoleate isotopomers featured coupling
3 days and another 3 days at 158C) were identical to those of acetylide synthons with the tosylates of enantioenriched
used previously in the study of calendate biosynthesis.[15] The (90–95% ee) [D1]alcohol intermediates[20] (Scheme 3); details
cellular fatty acid fractions were isolated from the harvested are given in the Supporting Information.
cells by hydrolysis/methylation and the deuterium content of
methyl a-eleostearate products was assessed by GC-MS. This
analysis showed that in both incubations, the product
consisted of a mixture of D0 and D1 species derived from D0
substrate and D2 substrate respectively (See Table 1).
Table 1: Intermolecular isotopic discrimination in the Aleurites fordii
Hemsl. D11,13-desaturase-mediated 1,4-desaturation of [11,11-
D2]linoleate and [14,14-D2]linoleate.
Linoleic acid
substrate
a-Eleostearic acid
KIE
product
%D0
%D2
%D0
%D1
C11 49.7ꢁ1.1 50.3ꢁ1.1 82.8ꢁ1.0 17.2ꢁ1.0 4.9ꢁ0.3[a]
C14 53.1ꢁ0.7 46.9ꢁ0.7 55.9ꢁ1.0 44.1ꢁ1.0 1.12ꢁ0.04[a]
[a] The standard deviation in KIE is based on the results of at least three
incubations.
Scheme 3. Synthesis of enantioenriched [D1]linoleates 1 from corre-
sponding [D1]alcohols. Asterisks denote the position of the D1-contain-
ing stereocenter.
The product kinetic isotope effects (kH/kD)[18] were
calculated as:
The saponified methyl esters of (8R)- and (8S)-[8-D1]-1,
(11R)- and (11S)-[8-D1]-1 were administered separately, in
triplicate, to growing cultures of the pYJ/INVSC1 strain of S.
cerevisiae (50 mL) expressing linoleate D8,10-desaturase (the
enzyme responsible for calendate formation).[15] The corre-
sponding experiment involved incubation of (11R)- and
kH % D0ðPÞ=% D1ðPÞ
ð1Þ
¼
kD
% D0ðSÞ=% D2ðSÞ
where P is product and S is substrate.
Using the formulation described above, the presence of (11S)-[8-D1]-1, (14R)- and (14S)-[11-D1]-1 with the pDR100/
a large primary deuterium isotope effect (4.9 ꢁ 0.3) for the INVSCI strain of S. cerevisiae that expresses the D11,13
-
carbon–hydrogen bond cleavage at C11 was calculated, desaturase enzyme forming a-eleostearate.[13] The incubation
whereas the carbon–hydrogen bond cleavage at C14 was conditions were identical to those used in the KIE experi-
found to be relatively insensitive to isotopic substitution ments (see above). The cellular fatty acid fractions were
(KIE = 1.12 ꢁ 0.04).
isolated by hydrolysis/methylation and analyzed by GC-MS to
The data presented above (that is, one large KIE at C11 determine the fate of the deuterium label in each case
and one small KIE at C14) clearly demonstrate that a- (Table 2).
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Table 2: Isotopic content of D1 substrates and D8,10- or D11,13-desaturated
products.
a switch in desaturase enantioselectivity involved a change in
substrate structure (D13-desaturation of an 11-yne versus 11-
ene in the moth T. pityocampa)[23] with concomitant alteration
in substrate conformation. In our case, an alteration in active
site geometry is presumably forcing a conformational change,
as will be discussed in more detail below.
D1 species
Substrates[a]
Products[a]
calendate a-eleo
stearate
%D0 %D1 %D0 %D1 %D0 %D1
The third FAD2 variant we analyzed is found in Dimor-
photheca sinuata (African daisy) and catalyzes the formation
of dimorphecolic acid ((S)-10E, 12E-9-hydroxyoctadecadie-
noic acid), a unique hydroxylated conjugated fatty acid.[24]
Molecular biological studies reveal that two FAD2-like
enzymes (DsFAD2-1 and DsFAD2-2) work in tandem fash-
ion to produce dimorphecolic acid from oleic acid by the 12E
isomer of linoleate as shown in Scheme 5.[8] By analogy with
(8R)-[8-D1]-1
(8S)-[8-D1]-1
(11S)-[11-D1]-1
(11R)-[11-D1]-1
(14S)-[14-D1]-1
(14R)-[14-D1]-1
1.2
1.1
0.8
2.5
1.3
1.2
98.8 73.2 26.8
98.9 9.6 90.4
99.2 61.4 38.6
8.2 91.8
97.5
98.7
98.8
5.1 94.9 74.9 25.1
10.7 89.3
94.3
5.7
[a] The average standard deviation in the percent isotopic content of
each species ꢁ0.4.
It is clear that calendate derived from (8R)-[8-D1]linoleic
acid and (11S)-[11-D1]linoleic acid proceeded with overall
loss[21] of deuterium during the enzymatic reaction. Deute-
rium was retained in the calendate product derived from (8S)-
[8-D1]linoleic acid and (11R)-[11-D1]linoleic acid. Precisely
the opposite trends were observed for a-eleostearate forma-
tion. That is, the majority[21] of the a-eleostearate sample
derived from (11R)-[11-D1]linoleic acid is nondeuterated,
whilst a-eleostearate product derived from (11S)-[11-
D1]linoleic acid is largely monodeuterated. a-Eleostearate
samples derived from (14R)-[14-D1]linoleic acid and (14S)-
[14-D1]linoleic acid were mainly nondeuterated (deuterium
loss) and monodeuterated (deuterium retention), respec-
tively. The results are summarized in Scheme 4.
Scheme 5. Biosynthesis of dimorphecolic acid.
calendate and a-eleostearate formation, we reasoned that
dimorphecolate would arise by regioselective hydroxy group
capture (SH2) by an allylic radical intermediate derived by
initial 11-H abstraction (Scheme 5). The anticipated syn
relationship between hydrogen removal and hydroxy group
rebound could be tested by incubating the two enantiomers of
11-[D1]oleate with a S. cerevisiae strain (DsFAD2-1,DsFAD2-
2/INVSc1; see the Supporting Information) co-expressing the
genes that encode for the DE12-desaturase (DsFAD2-1) and
Scheme 4. Stereochemistry of calendate and a-eleostearate formation.
Therefore, the facial selectivity of C8 and C11 hydrogen
removal during calendate formation is the same as that
observed for the parent D12-desaturase (FAD2) assuming an
extended substrate conformation in both cases (compare
Scheme 1 and 2). It is interesting to note that the enantiose-
lectivity of calendate formation is also identical to that
observed for an insect D10,12-desaturase;[22] this suggests that
the overall contours of the 1,4-desaturase binding site of these
enzymes are similar.
D
10,9OH-hydroxylase (DsFAD2-2).[8]
Methyl (11S)- and (11R)-[11-D1]octadec-9Z-enoate were
available as synthetic intermediates from the two mechanistic
studies described above (Scheme 3). These were incubated, in
triplicate, as the free acids with growing cultures of the
DsFAD2-1,DsFAD2-2/INVSc1 strain of S. cerevisiae (50 mL)
at 288C for 7 days. The cellular fatty acid fraction of the
harvested yeast cells were collected by hydrolysis/methylation
procedure and treated with BSTFA/pyridine to produce the
trimethylsilyl derivative of methyl dimorphecolate.
However the enantiofacial selectivity of hydrogen
removal for D11,13-desaturation is opposite to that D10,12
-
The isotopic content of the latter was evaluated by
desaturation (calendate formation) and indeed that of the
parent D12-desaturase (FAD2) and all other desaturases
studied to date, as has been summarized previously.[4,5] This
was an unexpected result. The only other reported case of
analyzing the base-peak ion cluster (M-157: ([Me-
+
=
ꢀ
=
ꢀ
ꢀ ꢀ
(CH2)4CH CH CH CH CH O Si(CH3)3] )) at m/z 225.
The molecular ion cluster of cellular oleate (m/z 296) and the
D
E12-desaturated intermediate ((m/z 294) was used to deter-
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Table 3: Isotopic content of cellular stereospecifically labeled mono-
deuterated oleates, 12E isomer of linoleate intermediate and dimor-
phecolate product isolated from S. cerevisiae strain, DsFAD2-1,DsFAD2-
2/INVSC1, expressing DE12-desaturase and D10,9OH-hydroxylase.
attack for the parent enzyme (C12 for FAD2 and C10 for
stearoyl-ACP D9-desaturase). This implies that the conforma-
tion of the substrates in the parent and variant/mutant in
these cases is probably very similar.
Substrate
Cellular
oleate[a]
12E isomer of Dimorphecolate[a]
In summary, we have shown how three homologous
FAD2-type enzymes complete an initial 11-H abstraction
event to give trienoic (calendate and a-eleostearate) and
hydroxylated dienoic (dimorphecolate) products with overall
syn-stereochemistry. To correlate these reactions with others
in the FAD2 family, one can postulate the existence of three
possible[26] substrate conformations (Scheme 6). As a 9Z
linoleate[a]
%D0 %D1 %D0 %D1 %D0
%D1
(11S)-[11-D1]-2
(11R)-[11-D1]-2
39.1 60.9 22.4
39.4 60.6 38.3
77.6
61.7
84.4
48.3
15.6
51.7
[a] The average standard deviation for each species is ꢁ1.6 (expressed
as a percentage).
mine the deuterium content of these compounds. The
analytical data is displayed in Table 3.
Dimorphecolate derived from (11S)-[11-D1]oleic acid has
a markedly lower percent D1 content than that derived from
the (R)-enantiomer. The high percent D0 content in both
samples could be traced to the contribution from endogenous
D0 oleate to the 9Z,12E-octadecadienoate substrate pool
(Table 3). The apparent stereoselectivity for loss of deuterium
from the proS C11 position is 80% [(100-100 (15.6/77.6)];
a value of 84% [(100(51.7/61.7)] was calculated for the
retention of deuterium from the pro R C11 position. The
operation of a large deuterium KIE in the removal of
hydrogen/deuterium from the pro S C11 position can be
inferred from the elevated D1/D0 ratio (77.6:22.4) of the 12-E-
isomer of linoleate (9Z,12E-octadecadienoate) intermediate
in the incubation of (11S)-[11-D1]-2 incubation compared to
that observed in the (11R)-[11-D1]-2 experiment (61.7:38.4;
Table 3). This implies that (11S)-[11-D1]-9Z,12E-octadeca-
dienoate reacts slower than (11R)-[11-D1]-9Z,12E-octadeca-
dienoate relative to endogenous [D0]oleate. This hypothesis
was tested by incubating a mixture of 11-[D2]oleate versus
oleate bearing a remote 18-[D3] label and evaluating the D1/
D3 ratio of the dimorphecolate product. An estimated value
of 7.8 ꢁ 0.8 for the KIE for C11H abstraction was obtained
(data not shown).
Scheme 6. Top: Prototypical substrate conformation for FAD2 parent
operating on oleate. Bottom: Three possible substrate conformations
for FAD2 variants operating on linoleate. *Dimorphecolate is derived
from the 12E isomer of linoleate.
The results of our stereochemical investigation confirm
our hypothesis that dimorphecolate biosynthesis involves H-
abstraction and hydroxy group rebound from the same side of
the olefinic plane. The enantioselectivity for 11-H abstraction
was the same as for calendate formation. This implies that
both enzymes utilize a similar substrate conformation; the
unusually high sequence similarity between the two enzymes
has been noted.[8] The switch that controls delivery of
a hydroxy group to C9 of the putative allylic radical
intermediate versus H-abstraction at C8 is probably purely
geometric given that both processes are expected to be highly
exergonic. The latent ability of desaturases to act as allylic
hydroxylators was revealed in a recent serendipitous discov-
ery by Shanklin and co-workers: a site-directed mutagenized,
triple mutant T117R/G188L/D280K of castor stearoyl-ACP
D9-desaturase oxidizes (Z)-9-octadecenoyl-ACP to produce
double bond is a necessary feature of all FAD2 substrates, the
simplifying assumption can be made that the position of the
C1–C10 portion is relatively fixed and that the C11–C18 half
of fatty acid is more mobile. The extended (quasi-anti)
conformation (A) is the most common and leads to calendate,
dimorphecolate, and vernolate formation by oxidative attack
from above the olefinic plane. It is presumably also the
conformation used in the parent FAD2-catalyzed reaction.
The energetically difficult, C12-initiated[11] dehydrogenation
of Z-alkenes to form an alkyne (crepenynate formation)
would proceed by abstraction of vinyl hydrogen atoms from
conformation B where the C12–C13 double bond is pointing
towards the oxidant. This conformation effectively blocks
competing oxidative pathways that are in principle energeti-
cally more favorable, such as H abstraction at C11. Finally we
have uncovered evidence for the presence of a third con-
formation (C) that appears to be involved in a-eleostearate
formation. It would be of interest to ascertain whether this
novel conformation is also involved in the formation of the
12E-isomer of linoleate from oleate[13] and the 13Z isomer of
a-eleostearate (punicic acid) from linoleate.[27] The topolog-
ꢀ
ꢀ
=
ꢀ
ꢀ
9-hydroxy-(10E)-octadecenoic acid ( CH2 CH CH CH2
[25]
ꢀ
=
ꢀ
ꢀ
ꢀ
! CH CH CH(OH) CH2 ). We find it significant that
for both the FAD2 variant (dimorphecolate formation) and
the Shanklin D9-desaturase mutant, allylic hydroxylation is
initiated at C11, the carbon atom proximal to the site of initial
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[15] D. W. Reed, C. K. Savile, X. Qiu, P. H. Buist, P. S. Covello, Eur. J.
Biochem. 2002, 269, 5024 – 5029.
[16] P. H. Buist, B. Behrouzian, J. Am. Chem. Soc. 1996, 118, 6295 –
6296.
ical considerations summarized above should play an essen-
tial part in completing our understanding of the factors
controlling reaction outcome. More precise information on
how the active site of each FAD2 variant is sculpted to
influence the course of oxidation must await structural
characterization of these membrane-bound enzymes.
[17] Methyl [11,11-D2]linoleate and [14,14-D2]linoleate were pre-
pared in straightforward fashion by alkynylation of the tosylate
of [1,1-D2]-2-octyn-1-ol or [4,4-D2]-2-octyn-1-ol with the acety-
lide salt of methyl 9-decynoate followed by Lindlar reduction of
the skipped diyne product (see the Supporting Information).
[18] L. Melander, W. H. Saunders, Jr., Reaction Rates of Isotopic
Molecules, 2nd ed., Wiley, Hoboken, 1979, pp. 91 – 129. The
contribution of a a secondary isotope by the spectator deuterium
atom in the D2-substrate is expected to be minor (10%).[16]
[19] L. Crombie, A. D. Heavers, J. Chem. Soc. Perkin Trans. 1 1992,
1929 – 1937.
Received: March 15, 2012
Published online: May 23, 2012
ꢀ
Keywords: C H activation · desaturases ·
enzyme mechanisms · kinetic isotope effects ·
conformation analysis
.
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