Hidden stereospecificity in the biosynthesis of divinyl
ether fatty acids
Mats Hamberg
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Sweden
Fatty acid hydroperoxides generated in plant tissues
by lipoxygenases or a-dioxygenases are subject to sec-
ondary modification by several enzymes [1]. One
branch of hydroperoxide metabolism involves forma-
tion of divinyl ether derivatives by carbon–carbon
bond cleavage catalyzed by specific divinyl ether synth-
ases. This type of transformation was first described in
1972 by Galliard and Phillips, who found that extracts
of potato tuber catalyzed the conversion of linoleic
acid 9(S)-hydroperoxide (9(S)-HPOD) into a divinyl
ether they named colneleic acid [2]. More recent work
has demonstrated that the divinyl ether synthase pro-
ducing colneleic acid and the related colnelenic acid is
induced in plant leaves during attack by fungal patho-
gens [3,4], and that divinyl ether fatty acids inhibit
mycelial growth and spore germination in certain fungi
Keywords
divinyl ether synthase; double bond
configuration; mechanism; stereospecifically
deuterated hydroperoxides; stereospecificity
of hydrogen abstraction
Correspondence
M. Hamberg, Department of Medical
Biochemistry and Biophysics, Division of
Physiological Chemistry II, Karolinska
Institutet, S-171 77 Stockholm, Sweden
Fax: +46 8736 0439

of the two hydrogen atoms a to the hydroperoxide carbon. Furthermore, a
consistent relationship between the absolute configuration of the hydrogen
atom eliminated (R or S) and the configuration of the introduced vinyl
ether double bond (E or Z) emerged from these results. Thus, irrespective
of which hydroperoxide regioisomer served as the substrate, divinyl ether
synthases abstracting the pro-R hydrogen generated divinyl ethers having
an E vinyl ether double bond, whereas enzymes abstracting the pro-S
hydrogen produced divinyl ethers having a Z vinyl ether double bond.
Abbreviations
colneleic acid, 9-[1¢(E),3¢(Z)-nonadienyloxy]-8(E)-nonenoic acid; colnelenic acid, 9-[1¢(E),3¢(Z),6¢(Z)-nonatrienyloxy]-8(E)-nonenoic acid; etheroleic
acid, 12-[1¢(E)-hexenyloxy]-9(Z),11(E)-dodecadienoic acid; etherolenic acid, 12-[1¢(E),3¢(Z)-hexadienyloxy]-9(Z),11(E)-dodecadienoic acid; 9(S)-
HPOD, 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid; 13(S)-HPOD, 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid.
736 FEBS Journal 272 (2005) 736–743 ª 2005 FEBS
[3,5]. Further research stimulated by these findings has
resulted in the cloning of genes encoding divinyl ether
synthases in tomato and potato and in the identifica-
tion of these enzymes as cytochrome P-450 proteins
[4,6].
The divinyl ether synthase-catalyzed formation of
colneleic acid from 9(S)-hydroperoxy-10(E),12(Z)-octa-
decadienoic acid in homogenate of potato tuber is
believed to take place via an enzyme-bound epoxide
carbocation [7–10]. Studies using stereospecifically deu-
terated substrate have shown that the double bond-
forming step in the biosynthesis of colneleic acid
involves stereoselective removal of the pro-R hydrogen
from C8 [8]. Several new divinyl ether synthases in
addition to the colneleic acid-forming enzyme have
been discovered during the last decade [11–14], and the
aim of this study was to determine the stereospecifici-

acid, i.e. 8(Z)-colneleic acid, was recently isolated from
leaves of the plant Clematis vitalba [14]. Interestingly,
biosynthesis of this compound from [8(R)-
2
H]9(S)-
HPOD took place with retention of the deuterium label,
i.e. a result opposite to that observed in the biosynthesis
of colneleic acid (Table 1, Fig. 2).
Stereospecificity of hydrogen eliminations from
C14 in the biosynthesis of etheroleic acid isomers
In the biosynthesis of etheroleic acid and its two iso-
mers, i.e. x5(Z)-etheroleic acid and 11(Z)-etheroleic
acid, one hydrogen is lost from C14. As seen in
Table 1, these conversions also took place with stereo-
specific hydrogen removals. In the case of etheroleic
acid (garlic) and 11(Z)-etheroleic acid (Ranunculus
Fig. 1. Reactions used to prepare stereospecifically deuterated fatty
acid hydroperoxides. The following reagents ⁄ treatments were used
(typical percentage yields are given within parenthesis): i, cinchoni-
dine (resolution; 10%); ii, acetyl chloride followed by treatment with
water ⁄ acetone (95%); iii, anodic coupling with methyl hydrogen
tridecanedioate (34%); iv, NaOH in methanol ⁄ water (95%); v,
CH
2
N
2
(99%); vi, p-toluenesulfonyl chloride ⁄ pyridine (86%); vii, lith-
ium aluminium deuteride (82%); viii, chromic acid (90%); ix, sodium
acetate (90%); x, Tetrahymena pyriformis (10%); xi, soybean lip-
oxygenase (95%). R

providing insights into reaction mechanisms, results
obtained with specifically labeled substrates have been
useful in molecular modeling studies of enzyme–sub-
strate complexes. An example from the oxylipin ⁄ eicos-
anoid field is the use of precursor acids labeled with
tritium in the 13(R) or 13(S) positions to elucidate the
stereochemistry and mechanism of the cyclooxygenase
reaction leading to prostaglandins [16], and the use of
this knowledge for establishing the productive confor-
mation of the arachidonic acid molecule bound to the
active site of the cyclooxygenase enzyme [18].
Divinyl ether synthases are cytochrome P-450
enzymes [4,6] and related to other (hydro)peroxide-
metabolizing P-450s such as allene oxide synthase [19],
hydroperoxide lyase [20,21], thromboxane synthase
[22] and prostacyclin synthase [22]. In the case of divi-
nyl ether biosynthesis, the initial step is believed to
consist of cleavage of the O-O bond of the hydroper-
oxide to provide an alkoxy radical, which undergoes
cyclization and one-electron oxidation into an epoxide
carbocation [23]. Enzyme-assisted removal of a pro-
ton a to the epoxide group and cleavage of the car-
bon-carbon single bond of the epoxide group provides
the final divinyl ether structure (Fig. 3). The nature of
the hydrogen-abstracting group in divinyl ether synth-
ases is unkown but may be either a basic amino acid
residue [7] or the strongly basic Fe
III
-OH group of the
P-450 heme [23].

Etheroleic acid 31.8 94
x5(Z)-Etheroleic acid 1.5 4
11(Z)-Etheroleic acid 33.3 98
[8(R)-
2
H]9(S)-HPOD 48.8 100
Colneleic acid 4.0 8
8(Z)-Colneleic acid 48.0 98
Fig. 2. Stereospecificities of five divinyl ether synthases. R
1
¼
(CH
2
)
6
-COOH, R
2
¼ (CH
2
)
7
-COOH. DES, divinyl ether synthase.
Fig. 3. Proposed sequence of reactions in the biosynthesis of divi-
nyl ether fatty acids. R
1
¼ C
5
H
11
and R

terms of the conformations of the carbon–carbon sin-
gle bond a to the epoxide of the epoxide carbocation
intermediate, i.e. transoid and cisoid conformations are
needed to produce E and Z vinyl ether double bonds,
respectively (Fig. 4). As seen from this model, irres-
pective of the detailed structure of the surrounding act-
ive site, rotation of the carbon–carbon single bond to
produce the two conformations moves either the pro-R
or pro-S hydrogen in contact with the same region of
the active site. This may be taken to suggest that the
positioning of the hydrogen-abstracting group relative
to the bound substrate is highly conserved in all divi-
nyl ether synthases of higher plants. The stereochemi-
cal data also show that the hydrogen eliminated
consistently has a syn relationship to the vicinal oxy-
gen atom (Fig. 4). This stereochemistry is in agreement
with the notion [23] that the heme iron not only parti-
cipates in the hydroperoxidase reaction but also serves
as the hydrogen-abstracting group. Further interpret-
ation of the stereochemical data presented must await
access of the three-dimensional structures of the divi-
nyl ether synthase P-450s.
Experimental procedures
Plant materials
Specimens of Ranunculus acris L., Ranunculus lingua L and
Clematis vitalba L. were obtained as described previously
[12–14]. Leaves were either used directly or shock-frozen in
liquid nitrogen and stored at )80 °C until use. Tubers of
potato (var. Bintje) and bulbs of garlic were obtained from
a local market.

+
SiMe
3
](m ⁄ z 311 and 312 in the undeuterated and
deuterated derivatives, respectively). As expected from the
localization of the deuterium atom at C8, the fragment
[(CH ¼ CH)
2
-CH(OSiMe
3
)-(CH
2
)
4
-CH
3
]
+
(m ⁄ z 225) was
devoid of deuterium.
3(R,S)-Hydroxyheptanoic acid
Methyl 3-oxoheptanoate (39.5 g; 0.25 mmol; Fluka Chemie
GmbH, Buchs, Switzerland) was dissolved in methanol
(250 mL) and sodium borohydride (4 g) was added at 0 °C
over a period of 3 h under magnetic stirring. Subsequently,
a solution of sodium hydroxide (12 g) in water (100 mL)
was added and the mixture was stirred for 15 h at 23 °C.
Extraction with diethyl ether provided 3(R,S)-hydroxyhept-
anoic acid (36.1 g; 99%) as a colorless viscous oil which
slowly solidified at room temperature. The purity as

4
H
9
and R
2
¼ (CH
2
)
7
-COOH (etheroleic acid series). ‘B’ attached
to the enzyme surfaces, base.
M. Hamberg Stereospecificity of divinyl ether synthases
FEBS Journal 272 (2005) 736–743 ª 2005 FEBS 739
mass that separated was collected on a Bu
¨
chner funnel and
redissolved in CCl
4
(700 mL). The solution was left over-
night at 23 °C and the crystals formed ( 20 g) were again
subjected to crystallization from CCl
4
. After six such crys-
tallizations, rosette-formed crystals (2.5 g) of the cinchoni-
dine salt of 3(R)-hydroxyheptanoic acid were obtained.
Regeneration of the acid by acidification and extraction
with diethyl ether provided 3(R)-hydroxyheptanoic acid
(0.73 g; yield, 10% of the theoretical) having [a]
D
23

ions was recorded: m ⁄ z 296 (M
+
– 18; loss of H
2
O), 283
(M
+
– 31; loss of OCH
3
), 257 (M
+
– 57; loss of (CH
2
)
3
-
CH
3
), 225 (257–32; loss of CH
3
OH), 185, 143, 87, and 69.
Methyl 14(R)-p-toluenesulfonyloxystearate
Methyl 14(R)-hydroxystearate (157 mg, 0.5 mmol) was dis-
solved in dry pyridine (4 mL), cooled to )25 °C and treated
with p-toluenesulfonyl chloride (400 mg). After 12 h at
)25 °C and 48 h at +4 °C, water was added and the solu-
tion was extracted with diethyl ether. Purification by open
column silicic acid chromatography afforded methyl 14(R)-
p-toluenesulfonyloxystearate (200 mg, yield, 86%).
[14(S)-

143, 87, and 74.
[14(S)-
2
H]Linoleic acid
A culture of Tetrahymena pyriformis strain phenoset A
(American Type Culture Collection #30327; Manassas, VA,
USA) was added to culture medium (400 mL) consisting of
glucose (0.5%, w ⁄ v), yeast extract (0.5%, w ⁄ v) and peptone
(0.5%, w ⁄ v) in 0.004 m potassium phosphate buffer pH 7.0
and containing the sodium salt of [14(S)-
2
H]stearic acid
(10 mg). The mixture was incubated under continuous sha-
king at 32 °C for 92 h [8]. The cell pellet collected by cen-
trifugation was suspended in 50% aqueous methanol
(100 mL) containing sodium hydroxide (7 g) and the mix-
ture was refluxed under an atmosphere of argon for
90 min. The isolated mixed fatty acids ( 9 mg) were
subjected to semipreparative RP-HPLC using a column of
Nucleosil C
18
100-7 (250 · 10 mm) purchased from
Macherey-Nagel (Du
¨
ren, Germany) and a solvent system
of acetonitrile ⁄ water ⁄ acetic acid (800 : 200 : 0.1, v ⁄ v ⁄ v) at
3mLÆmin
)1
. [14(S)-
2

ions due to the fragment [(CH ¼ CH)
2
-CH(OSiMe
3
)-
(CH
2
)
4
-CH
3
]
+
(m ⁄ z 225 and 226 in undeuterated and
Stereospecificity of divinyl ether synthases M. Hamberg
740 FEBS Journal 272 (2005) 736–743 ª 2005 FEBS
deuterated hydroxides, respectively). The isotopic composi-
tion found after correction for the natural abundance of
the m ⁄ z 226 ion was 33.9% monodeuterated and 66.1% un-
deuterated molecules. As expected from the localization of
the deuterium label at C14, the fragment CH
3
OOC-(CH
2
)
7
-
(CH ¼ CH)
2
-CH ¼ O

2
H]linoleic
acid (4 mg; ratio of labeled ⁄ unlabeled molecules, 0.62).
[14(R)-
2
H]13(S)-Hydroperoxy-9(Z),11(E)-octadecadi-
enoic acid
[14(R)-
2
H]Linoleic acid (4 mg) was incubated with soybean
lipoxygenase as described above. Following purification by
silicic acid open column chromatography, [14(R)-
2
H]13(S)-
hydroperoxy-9(Z),11(E)-octadecadienoic acid (3 mg) was
obtained. The isotopic composition as determined by
GC ⁄ MS analysis of the methyl ester⁄ Me
3
Si derivative of
the reduced compound was 38.3% monodeuterated and
61.7% undeuterated molecules.
Enzyme preparations
The following divinyl ether synthase preparations were used
for study of the biosynthesis of the divinyl ethers indicated.
Colneleic acid
Tubers of potato were sliced and homogenized at 0 °Cin
0.1 m borate buffer pH 9.0 (2 : 1, v ⁄ w) using an Ultra-Tur-
rax. The homogenate was filtered through gauze and centri-
fuged at 9300 g for 15 min. Further centrifugation at
105 000 g provided a particulate fraction which was resus-

for 20 min with 300 lm [14(R)-
2
H]- or [14(S)-
2
H]13(S)-
HPOD. In the same way, filtered homogenates of C. vitalba
(45 mL) or suspensions of the 105 000 g fraction of potato
tuber homogenate were stirred with 300 lm [8(R)-
2
H]
9(S)-HPOD. Material obtained after extraction with diethyl
ether was subjected to solid-phase extraction using an amino-
propyl column (0.5 g; Supelco, Bellefonte, PA, USA) [12].
Material eluted with diethyl ether ⁄ acetic acid (98 : 2, v ⁄ v)
was esterified by treatment with diazomethane and subjected
to RP-HPLC using a column of Nucleosil C
18
100–7
(250 · 10 mm) and a solvent system of acetonitrile ⁄ water
(80 : 20, v ⁄ v) at a flow rate of 4 mLÆmin
)1
. The effluent was
led to a Bischoff model DAD-100 diode-array detector (Bis-
choff Chromatography, Leonberg, Germany), and divinyl
M. Hamberg Stereospecificity of divinyl ether synthases
FEBS Journal 272 (2005) 736–743 ª 2005 FEBS 741
ethers localized by their strong absorption at 250–253 nm
were collected, esterified, and analyzed for deuterium content
by GC ⁄ MS. Blank incubations were performed in which the
divinyl ether synthase preparations were incubated in the

H]9(S)-
HPOD; reduced, methyl esterified and trimethylsilylated),
and m ⁄ z 308 and 309 (methyl esters of divinyl ether fatty
acids).
Acknowledgements
Mrs Gunvor Hamberg is thanked for expert technical
assistance and for collection and identification of the
plant materials used. This work was supported by a
generous grant given by the late Professor Sune Bergs-
tro
¨
m, Stockholm, and by grants from the Swedish
Research. Council for Environment, Agricultural
Sciences and Spatial Planning (project no. 2001-2553)
and the European Union (project No. QLK5-CT-2001-
02445).
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