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 9S-hydroperoxide 9S-HPO
Trang 1ether 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
Tel: +46 852487640
E-mail: Mats.Hamberg@mbb.ki.se
(Received 13 October 2004, revised 29
November 2004, accepted 1 December
2004)
doi:10.1111/j.1742-4658.2004.04510.x
Incubations of [8(R)-2H]9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid, [14(R)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid and [14(S)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid were per-formed with preparations of plant tissues containing divinyl ether synth-ases In agreement with previous studies, generation of colneleic acid from the 8(R)-deuterated 9(S)-hydroperoxide was accompanied by loss of most
of the deuterium label (retention, 8%), however, the opposite result (98% retention) was observed in the generation of 8(Z)-colneleic acid from the same hydroperoxide Formation of etheroleic acid and 11(Z)-etheroleic acid from the 14(R)-deuterated 13(S)-hydroperoxide was accompanied by loss
of most of the deuterium (retention, 7–8%), and, as expected, biosynthesis
of these divinyl ethers from the corresponding 14(S)-deuterated hydroper-oxide was accompanied by retention of deuterium (retention, 94–98%) Biosynthesis of x5(Z)-etheroleic acid from the 14(R)- and 14(S)-deuterated 13(S)-hydroperoxides showed the opposite results, i.e 98% retention and 4% retention, respectively The experiments demonstrated that biosynthesis
of divinyl ether fatty acids from linoleic acid 9- and 13-hydroperoxides takes place by a mechanism that involves stereospecific abstraction of one
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)-9(S)-HPOD, 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid.
Trang 2[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-ties of these additional enzymes using hydroperoxides
stereospecifically deuterated at the appropriate carbon
atoms
Results
Stereospecifically deuterated fatty acids and
hydroperoxides
Stereospecifically deuterated stearates were synthesized
starting with 8- or 14-hydroxystearates of high optical
purity Deuterium was introduced by reduction of the
corresponding p-toluenesulfonates with lithium
alumin-ium deuteride, a reaction that takes place with clean
inversion of configuration [15] (Fig 1) The deuterated
stearates were converted to the corresponding
linole-ates by exposure to growing cultures of the flagellate
Tetrahymena pyriformis This biological desaturation
proceeds without significant degradation of the carbon
chain and without migration of the isotope [16],
how-ever, considerable dilution with unlabeled material
took place Incubation of the deuterated linoleates
with soybean lipoxygenase or tomato lipoxygenase
resulted in deuterated 13(S)-HPOD and 9(S)-HPOD,
respectively, which had a deuterium content of 34–
49% (Table 1)
Stereospecificity of hydrogen eliminations from
C8 in the biosynthesis of colneleic acid isomers
It has been shown previously that biosynthesis of
colne-leic acid from 9(S)-HPOD catalyzed by divinyl ether
synthase in potato tuber is accompanied by
stereoselec-tive loss of the pro-R hydrogen from C8 [8] This result
was confirmed here (Table 1) An isomer of colneleic
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 aluminlith-ium deuteride (82%); viii, chromic acid (90%); ix, sodlith-ium acetate (90%); x, Tetrahymena pyriformis (10%); xi, soybean lip-oxygenase (95%) R 1 ¼ (CH 2 ) 7 -COOCH 3 , R 2 ¼ (CH 2 ) 7 -COOH.
Trang 3lingua) an essentially complete loss of isotope was
noted with the 14(R)-deuterated precursor, whereas
incubation of the 14(S)-deuterated precursor afforded
products that retained most of the deuterium label
The opposite labeling pattern was observed in the
bio-synthesis of x5(Z)-etheroleic acid (Ranunculus acris),
i.e stereospecific removal of the pro-S hydrogen and
retention of the pro-R hydrogen (Table 1, Fig 2)
Discussion
Since the pioneering work by Schroepfer and Bloch on
the stereochemistry of the desaturation of stearate into
oleate [17], studies using stereospecifically deuterated
or tritiated precursors have generated important infor-mation on enzyme-catalyzed reactions In addition to 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 FeIII-OH group of the P-450 heme [23]
This study confirms and extends previous work on the divinyl ether synthase class of cytochrome P-450s
by showing that removal of one of the two hydro-gens a to the hydroperoxide group invariably takes place in a stereospecific way (Fig 2) Furthermore, inspection of the data given in Table 1 and Fig 2
Table 1 Isotope composition of deuterated fatty acid
hydroperox-ides and divinyl ether fatty acids Isotopic analysis was carried out
with selected ion monitoring mass spectrometry using the ions
m ⁄ z 225 and 226 (reduced[14– 2 H]13(S)-HPOD), m ⁄ z 311 and 312
(reduced[8- 2 H]9(S)-HPOD), and m ⁄ z 308 and 309 (divinyl ether fatty
acids).
Compound
Monodeuterated molecules (%)
Retention of deuterium (%)
Fig 2 Stereospecificities of five divinyl ether synthases R1¼
(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 R1¼ C 5 H11and R2¼ (CH 2 )6-COOH (colneleic acid series), or R 1 ¼ (CH 2 ) 7 -COOH and R 2 ¼ C 4 H 9 (etheroleic acid series) Adapted from Grechkin [23].
Trang 4reveals an interesting correlation, i.e biosynthesis of
divinyl ethers having the E configuration of the newly
created vinyl ether double bond (colneleic acid,
ethero-leic acid and 11(Z)-etheroethero-leic acid) takes place with
stereoselective loss of the pro-R hydrogen, whereas the
Zvinyl ethers (8(Z)-colneleic acid and x5(Z)-etheroleic
acid) are formed in a process which involves loss of
the pro-S hydrogen These results can be intepreted in
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
Stereospecifically deuterated hydroperoxides [8(R)-2H]9(S)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid
9(S)-HPOD stereospecifically labeled with deuterium in the 8(R) position was prepared via [8(S)-2H]stearic acid (25 mg) and [8(R)-2H]linoleic acid as described previously [8] An aliquot of the hydroperoxide was reduced by treatment with sodium borohydride in methanol at 0C, and the methyl ester⁄ Me3Si derivative of the resulting hydroxide was analyzed by GC⁄ MS The isotopic composition of the sample was 48.8% monodeuterated and 51.2% undeute-rated molecules as determined by mass spectrometric analysis
of the fragment [CH3OOC-(CH2)7-(CH¼ CH)2-CH¼
O+SiMe3] (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 [(CH¼ CH)2-CH(OSiMe3)-(CH2)4-CH3]+ (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 0C 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 23C 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 checked by GC⁄ MS analysis of the methyl ester ⁄ Me3Si derivative was > 95%
3(R)-Hydroxyheptanoic acid 3(R,S)-Hydroxyheptanoic acid (14.6 g, 0.1 mmol) and cinchonidine (29.4 g, 0.1 mmol) in carbon tetrachloride (1 L) was heated on a boiling water bath for 3 min and then allowed to cool to room temperature The crystalline
Fig 4 Conformations and hydrogen abstractions in the final step
of divinyl ether fatty acid biosynthesis (A) Divinyl ether synthases
from potato, garlic or Ranunculus lingua introducing an ‘E’ vinyl
ether double bond, (B) divinyl ether synthases from Ranunculus
acris or Clematis vitalba introducing a ‘Z ’ vinyl ether double bond.
R1¼ (CH 2 )6-COOH and R2¼ C 5 H11(colneleic acid series) or R1¼
C4H9and R2¼ (CH 2 )7-COOH (etheroleic acid series) ‘B’ attached
to the enzyme surfaces, base.
Trang 5mass that separated was collected on a Bu¨chner funnel and
redissolved in CCl4 (700 mL) The solution was left
over-night at 23C and the crystals formed ( 20 g) were again
subjected to crystallization from CCl4 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]D23¼
)23.3 (c 2.5, chloroform); earlier published for
3(R)-hydroxyhexanoic acid, [a]D¼)28 [24] and for
3(R)-hydroxyoctanoic acid, [a]D¼)21 [25] An aliquot was
esterified with diazomethane and converted to its
2(S)-phenylpropionyl derivative [26] Analysis by GLC showed
an enantiomeric composition of > 98% 3(R)- and < 2%
3(S)-hydroxyheptanoic acid
Methyl 14(R)-hydroxystearate
3(R)-Hydroxyheptanoic acid (438 mg, 3 mmol) was refluxed
for 15 min with acetyl chloride (20 mL) The residue
obtained following evaporation of the reagent was dissolved
in acetone (20 mL), and water (13.3 mL) was added under
magnetic stirring After 5 h at 23C, the solution was
extracted providing essentially pure 3(R)-acetoxyheptanoic
acid This material was dissolved in methanol (100 mL)
containing methyl hydrogen tridecane-1,13-dioate (5.16 g,
20 mmol) and sodium methoxide (1.2 mmol) The solution
was transferred to an electrolysis cell and a current of
1.8 A was passed through for 2 h The resulting product
was saponified, esterified by treatment with diazomethane,
and subjected to silicic acid open column chromatography
Elution with diethyl ether⁄ hexane (20 : 80, v ⁄ v) afforded
methyl 14(R)-hydroxyoctadecanoate (299 mg; yield, 32%)
Analysis by GC⁄ MS showed a single peak (purity, > 96%)
on which a mass spectrum showing the following prominent
ions was recorded: m⁄ z 296 (M+ – 18; loss of H2O), 283
(M+ – 31; loss of OCH3), 257 (M+ – 57; loss of (CH2)3
-CH3), 225 (257–32; loss of CH3OH), 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 Purificasolu-tion by open
column silicic acid chromatography afforded methyl
14(R)-p-toluenesulfonyloxystearate (200 mg, yield, 86%)
[14(S)-2H]Stearic acid
Methyl 14(R)-p-toluenesulfonyloxystearate (100 mg, 0.21
mmol) was refluxed with lithium aluminium deuteride
(300 mg; 98 atom percentage deuterium; purchased from Sigma-Aldrich) in dry tetrahydrofuran (25 mL) for 18 h The resulting octadecanol was dissolved in 6 mL of acetone and treated with 0.63 mL of Jones’ reagent at 23C for
30 min The product was purified by open column silicic acid chromatography to afford [14(S)-2H]stearic acid (44 mg, yield, 74%) Analysis of an aliquot (methyl ester)
by GC⁄ MS showed a single peak The mass spectrum showed the following prominent ions: m⁄ z 299 (M+
), 256 (M+ – 43; loss of C3H7), 200 (M+ – 99; loss of C7H15),
143, 87, and 74
[14(S)-2H]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)-2H]stearic acid (10 mg) The mixture was incubated under continuous sha-king at 32C 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 C18 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
3 mLÆmin)1 [14(S)-2H]Linoleic acid (2 mg) was collected at 68–72 mL effluent The mass spectrum of the methyl ester
of this material showed molecular ions at m⁄ z 294 and 295 corresponding to undeuterated and monodeuterated mole-cules, respectively (ratio, 0.51), the undeuterated linoleate being derived from the organism used to carry out the desaturation
[14(S)-2 H]13(S)-Hydroperoxy-9(Z),11(E)-octadecadi-enoic acid
[14(S)-2H]Linoleic acid (1.9 mg) was stirred for 10 min at
0C in 0.1 m sodium borate buffer pH 10.4 containing soy-bean lipoxygenase (soysoy-bean lipoxygenase type IV purchased from Sigma-Aldrich, 15 lL) Material isolated by extraction with diethyl ether was purified by silicic acid open column chromatography to provide 1.7 mg (yield, 80%) of the title compound showing kmax (EtOH)¼ 233 nm An aliquot was reduced with NaBH4 in methanol at 0C and the resulting deuterated 13-hydroxy-9(Z),11(E)-octadecadieno-ate was analyzed as the methyl ester⁄ Me3Si derivative by
GC⁄ MS using selected ion monitoring of the mass spectral ions due to the fragment [(CH¼ CH)2-CH(OSiMe3 )-(CH2)4-CH3]+ (m⁄ z 225 and 226 in undeuterated and
Trang 6deuterated hydroxides, respectively) The isotopic
composi-tion found after correccomposi-tion 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 CH3OOC-(CH2)7
-(CH¼ CH)2-CH¼ O+SiMe3 was undeuterated and gave
rise to an ion at m⁄ z 311 with insignificant enrichment of
m⁄ z 312
[14(R)-2H]Stearic acid
Methyl 14(R)-p-toluenesulfonyloxystearate (100 mg, 0.21
mmol) was dissolved in acetic acid (7 mL) containing
sodium acetate (50 mg) and the mixture was kept at 60C
for 17 h The product was saponified by refluxing with
10% NaOH in 80% aqueous methanol for 90 min and then
esterified by treatment with diazomethane and treated with
p-toluenesulfonyl chloride in pyridine as described above
Reduction with lithium aluminium deuteride followed by
Jones oxidation and silicic acid open column
chromato-graphy afforded [14(R)-2H]stearic acid (22 mg; yield, 37%)
(Fig 1)
[14(R)-2H]Linoleic acid
[14(R)-2H]Stearic acid (22 mg) was incubated with
Tetra-hymena pyriformis as described above and the free fatty
acids subjected to RP-HPLC to provide [14(R)-2H]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)-2H]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 ⁄ Me3Si 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 0C in
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-pended in borate buffer (half the volume compared with the corresponding 105 000 g supernatant) [8]
Etheroleic acid Bulbs of garlic were sliced and homogenized at 0C in 0.1 m potassium phosphate buffer pH 8.0 (2 : 1, v⁄ w) The homogenate was filtered through gauze and successively centrifuged at 1100 g for 15 min and 105 000 g to provide
a particulate fraction which was resuspended in phosphate buffer (half the volume compared with the corresponding
105 000 g supernatant) [11]
x5(Z)-Etheroleic acid
Leaves of Ranunculus acris were minced and homogenized
at 0C in 0.1 m potassium phosphate buffer pH 6.7 (5 : 1,
v⁄ w; buffer supplemented with 235 lm of salicylhydroxamic acid as an lipoxygenase inhibitor) The filtered homogenate was centrifuged at 1100 g for 15 min, and further centrifu-gation of the supernatant at 105 000 g provided a particu-late fraction that was resuspended in phosphate buffer (half the volume compared with the corresponding supernatant) [12]
11(Z)-Etheroleic acid Leaves of Ranunculus lingua were treated in the same way
as described for Ranunculus acris [13]
8(Z)-Colneleic acid Leaves of Clematis vitalba were homogenized at 0C in potassium phosphate buffer pH 6.7 (5 : 1, v⁄ w) and the homogenate was filtered through gauze [14]
Incubations and isolation of products Suspensions of the particulate fractions of homogenates of garlic, R acris or R lingua (2–20 mL) were stirred at 23C for 20 min with 300 lm [14(R)-2H]- 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)-2H] 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 C18 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
Trang 7ethers 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
absence of hydroperoxide and subsequently carried through
the whole sequence In the case of the enzyme preparation
obtained from garlic bulbs, significant amounts of
endo-genous etheroleic acid were detected, i.e 3–4% of the levels
achieved in incubations carried out in the presence of added
13(S)-HPOD The isotopic compositions of etheroleic acid
biosynthesized from deuterated precursors using this enzyme
preparation were corrected for the dilution caused by this
endogenous material By contrast, no significant occurrence
of endogenous divinyl ethers was detected in the other four
preparations
GC/MS
GC⁄ MS was carried out with a Hewlett-Packard model
5970B mass-selective detector connected to a
Hewlett-Pack-ard model 5890 gas chromatograph equipped with a
capil-lary column of 5% phenylmethylsiloxane (12 m, 0.33 lm
film thickness) Helium was used as the carrier gas, and the
column temperature was raised at 10CÆmin)1from 120 to
260C Isotopic composition of analytes were determined
using the selected ion monitoring mode and the following
mass spectral ions: m⁄ z 225 and 226 (undeuterated and
monodeuterated [14-2H]13(S)-HPOD; reduced, methyl
este-rified and trimethylsilylated), m⁄ z 311 and 312 ([8-2
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)
References
1 Feussner I & Wasternack C (2002) The lipoxygenase
pathway Annu Rev Plant Biol 53, 275–297
2 Galliard T & Phillips DR (1972) The enzymic
conver-sion of linoleic acid into
9-(nona-1¢,3¢-dienoxy)non-8-enoic acid, a novel unsaturated ether derivative
isolated from homogenates of Solanum tuberosum
tubers Biochem J 129, 743–753
3 Weber H, Che´telat A, Caldelari D & Farmer EE (1999) Divinyl ether fatty acid synthesis in late blight-diseased potato leaves Plant Cell 11, 485–493
4 Stumpe M, Kandzia R, Go¨bel C, Rosahl S & Feussner
I (2001) A pathogen-inducible divinyl ether synthase (CYP74D) from elicitor-treated potato suspension cells FEBS Lett 507, 371–376
5 Grane´r G, Hamberg M & Meijer J (2003) Screening of oxylipins for control of oilseed rape (Brassica napus) fungal pathogens Phytochemistry 63, 89–95
6 Itoh A & Howe GA (2001) Molecular cloning of a divi-nyl ether synthase Identification as a CYP74 cyto-chrome P-450 J Biol Chem 276, 3620–3627
7 Crombie L, Morgan DO & Smith EH (1987) The enzy-mic formation of colneleic acid, a divinyl ether fatty acid: experiments with [(9S)-18O2 ]hydroperoxyoctadeca-(10E),(12Z)-dienoic acid J Chem Soc Chem Commun 502–503
8 Fahlstadius P & Hamberg M (1990) Stereospecific removal of the pro-R hydrogen at C-8 of 9(S)-hydroper-oxyoctadecadienoic acid in the biosynthesis of colneleic acid J Chem Soc Perkin Trans 1, 2027–2030
9 Corey EJ, Nagata R & Wright SW (1987) Biomimetic total synthesis of colneleic acid and its function as a lipoxygenase inhibitor Tetrahedron Lett 28, 4917– 4920
10 Gerwick WH (1996) Epoxy allylic carbocations as con-ceptual intermediates in the biogenesis of diverse marine oxylipins Lipids 31, 1215–1231
11 Grechkin AN, Fazliev FN & Mukhtarova LS (1995) The lipoxygenase pathway in garlic (Allium sativum L.) bulbs: detection of the novel divinyl ether oxylipins FEBS Lett 371, 159–162
12 Hamberg M (1998) A pathway for biosynthesis of divi-nyl ether fatty acids in green leaves Lipids 33, 1061– 1071
13 Hamberg M (2002) Biosynthesis of new divinyl ether oxylipins in Ranunculus plants Lipids 37, 427–433
14 Hamberg M (2004) Isolation and structures of two divi-nyl ether fatty acids from Clematis vitalba Lipids 39, 565–569
15 Helmkamp GK & Rickborn BF (1957) Stereochemistry
of some lithium aluminium deuteride reductions J Org Chem 22, 479–482
16 Hamberg M & Samuelsson B (1967) On the mechanism
of the biosynthesis of prostaglandins E1and F1a J Biol Chem 242, 5336–5343
17 Schroepfer GJ & Bloch K (1965) The stereospecific con-version of stearic acid to oleic acid J Biol Chem 240, 54–63
18 Malkowski MG, Ginell SL, Smith WL & Garavito RM (2000) The productive conformation of arachidonic acid bound to prostaglandin synthase Science 289, 1933– 1937
Trang 819 Song W-C & Brash AR (1991) Purification of an allene
oxide synthase and identification of the enzyme as a
cytochrome P-450 Science 253, 781–784
20 Matsui K, Shibutani M, Hase T & Kajiwara T (1996)
Bell pepper fruit fatty acid hydroperoxide lyase is a
cytochrome P-450 (CYP74B) FEBS Lett 394, 21–24
21 Grechkin AN & Hamberg M (2004) The ‘heterolytic
hydroperoxide lyase’ is an isomerase producing a
short-lived hemiacetal Biochim Biophys Acta 1636, 47–58
22 Hecker M & Ullrich V (1989) On the mechanism of
prostacyclin and thromboxane A2biosynthesis J Biol
Chem 264, 141–150
23 Grechkin AN (2002) Hydroperoxide lyase and divinyl ether synthase Prostaglandins Other Lipid Med 68–69, 457–470
24 Serck-Hanssen K (1956) Optically active higher aliphatic hydroxy-compounds Arkiv Kemi 10, 135–149
25 Lemieux RU & Giguere J (1951) Biochemistry of ustal-gines Can J Chem 29, 678–690
26 Hammarstro¨m S & Hamberg M (1973) Steric analysis
of 3-, x4-, x3- and x2-hydroxy acids and various alkan-ols by gas–liquid chromatography Anal Biochem 52, 169–179