Formation of a cyclopropyl epoxide via a leukotriene A synthase-related pathway in an anaerobic reaction of soybean lipoxygenase-1 with 15S-hydroperoxyeicosatetraenoic acid: Evidence that oxygen access is a determinant of secondary reactions with fatty acid hydroperoxides

Article abstract of DOI:10.1074/jbc.M109.084632

The further conversion of an arachidonic acid hydroperoxide to a leukotriene A (LTA) type epoxide by specific lipoxygenase (LOX) enzymes constitutes a key step in inflammatory mediator biosynthesis. Whereas mammalian 5-LOX transforms its primary product (5S-hydroperoxyeicosatetraenoic acid; 5S-HPETE) almost exclusively to LTA₄, the model enzyme, soybean LOX-1, normally produces no detectable leukotrienes and instead further oxygenates its primary product 15S-HPETE to 5,15- and 8,15-dihydroperoxides. Mammalian 15-LOX-1 displays both types of activity. We reasoned that availability of molecular oxygen within the LOX active site favors oxygenation, whereas lack of O₂ promotes LTA epoxide synthesis. To test this, we reacted 15S-HPETE with soybean LOX-1 under anaerobic conditions and identified the products by high pressure liquid chromatography, UV, mass spectrometry, and NMR. Among the products, we identified a pair of 8,15-dihydroxy diastereomers with all-trans-conjugated trienes that incorporated ¹⁸O from H ₂¹⁸O at C-8, indicative of the formation of 14,15-LTA4. A pair of 5,15-dihydroxy diastereomers containing two trans,trans-conjugated dienes (6E,8E,11E,13E) and that incorporated ¹⁸O from H ₂¹⁸O at C-5 was deduced to arise from hydrolysis of a novel epoxide containing a cyclopropyl ring, 14,15-epoxy-[9,10,11-cyclopropyl]- eicosa-5Z,7E,13E-trienoic acid. Also identified was the δ-lactone of the 5,15-diol, a derivative that exhibited no ¹⁸O incorporation due to its formation by intramolecular reaction of the carboxyl anion with the proposed epoxide intermediate. Our results support a model in which access to molecular oxygen within the active site directs the outcome from competing pathways in the secondary reactions of lipoxygenases.

Full text of DOI:10.1074/jbc.M109.084632

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 18, pp. 13427–13436, April 30, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Formation of a Cyclopropyl Epoxide via a Leukotriene A
Synthase-related Pathway in an Anaerobic Reaction
of Soybean Lipoxygenase-1 with
15S-Hydroperoxyeicosatetraenoic Acid
EVIDENCE THAT OXYGEN ACCESS IS A DETERMINANT OF SECONDARY REACTIONS WITH
□
S
FATTY ACID HYDROPEROXIDES*
Received for publication, November 12, 2009, and in revised form, January 29, 2010 Published, JBC Papers in Press, March 1, 2010, DOI 10.1074/jbc.M109.084632
Yuxiang Zheng and Alan R. Brash1
From the Department of Pharmacology and the Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232
Lipoxygenases (LOX)2 are named for their oxygenase activity
with polyunsaturated fatty acids, yet they also catalyze other
types of reaction. One of the most important in a biological
The further conversion of an arachidonic acid hydroperox-
ide to a leukotriene A (LTA) type epoxide by specific lipoxy-
genase (LOX) enzymes constitutes a key step in inflammatory
mediator biosynthesis. Whereas mammalian 5-LOX trans- context is leukocyte 5-LOX-catalyzed further conversion of the
hydroperoxide product 5S-HPETE to leukotriene A4 (LTA4)
(1). LTA4, an unstable epoxide, will then be subject to metabo-
lism of downstream enzymes in the pathway to more stable and
biologically active LTs, including the dihydroxy LTB4 and the
peptidyl LTC4, LTD4, and LTE4 (2). Parallels of the LTA syn-
thase activity of 5-LOX also exist in some other LOX enzymes.
Mammalian 15-LOX-1, for example, can further convert 15S-
HPETE to a structural analogue of LTA4, named 14,15-LTA4
based on the position of the epoxide group between C-14 and
C-15 (3, 4). This 14,15-epoxide was recently given the name of
eoxin A4 on account of its production by eosinophils and its
further transformation, analogous to LTA4, to a series of bio-
logically active peptidyl derivatives, 14,15-LTC4, -LTD4, and
-LTE4 (eoxins C4, D4, and E4) (5).
forms its primary product (5S-hydroperoxyeicosatetraenoic
acid; 5S-HPETE) almost exclusively to LTA4, the model
enzyme, soybean LOX-1, normally produces no detectable
leukotrienes and instead further oxygenates its primary prod-
uct 15S-HPETE to 5,15- and 8,15-dihydroperoxides. Mam-
malian 15-LOX-1 displays both types of activity. We reasoned
that availability of molecular oxygen within the LOX active
site favors oxygenation, whereas lack of O2 promotes LTA
epoxide synthesis. To test this, we reacted 15S-HPETE with
soybean LOX-1 under anaerobic conditions and identified
the products by high pressure liquid chromatography, UV,
mass spectrometry, and NMR. Among the products, we iden-
tified a pair of 8,15-dihydroxy diastereomers with all-trans-
conjugated trienes that incorporated 18O from H218O at C-8,
indicative of the formation of 14,15-LTA4. A pair of 5,15-
dihydroxy diastereomers containing two trans,trans-conju-
gated dienes (6E,8E,11E,13E) and that incorporated 18O from
H218O at C-5 was deduced to arise from hydrolysis of a novel
epoxide containing a cyclopropyl ring, 14,15-epoxy-[9,10,11-
cyclopropyl]-eicosa-5Z,7E,13E-trienoic acid. Also identified
was the ␦-lactone of the 5,15-diol, a derivative that exhibited
no 18O incorporation due to its formation by intramolecular
reaction of the carboxyl anion with the proposed epoxide
intermediate. Our results support a model in which access to
molecular oxygen within the active site directs the outcome
from competing pathways in the secondary reactions of
lipoxygenases.
Despite the general parallels between leukocyte 5-LOX and
mammalian 15-LOX-1 within the physiological context, the
two enzymes exhibit notable differences in their reactions
toward 5S-HPETE and 15S-HPETE, respectively. Leukocyte
5-LOX gives almost exclusively LTA4 from 5S-HPETE,
whereas the reaction of mammalian 15-LOX-1 with 15S-
HPETE leads, in addition to 14,15-LTA4, to several other prod-
ucts, including two fatty acid dihydroperoxides, 8S,15S-
diHPETE and 5S,15S-diHPETE (3). On the other end of the
spectrum of reactivity compared with leukocyte 5-LOX is the
prototypical plant lipoxygenase, soybean LOX-1. It converts
15S-HPETE exclusively to 8S,15S-diHPETE and 5S,15S-
diHPETE (6). These diHPETEs are often referred to as “double
oxygenation” products because they are formed, in essence, by
oxygenation of arachidonic acid twice in succession at two dif-
ferent positions.
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth 2 The abbreviations used are: LOX, lipoxygenase; LT, leukotriene; HPODE,
Grant AR-051968 (to A. R. B.).
hydroperoxyoctadecadienoic acid; HPETE, hydroperoxyeicosatetraenoic
acid; HETE, hydroxyeicosatetraenoic acid; diH(P)ETE, dihydro(pero)xyeico-
satetraenoic acid; HPLC, high pressure liquid chromatography; RP,
reversed phase; SP, straight phase; GC, gas chromatography; MS, mass
spectrometry; LC, liquid chromatography; ESI, electrospray ionization;
NDGA, nordihydroguaiaretic acid; TMS, trimethylsilyl; KETE, ketoeicosatet-
raenoic acid.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S15, Tables S1–S3, and additional references.
1 To whom correspondence should be addressed: Dept. of Pharmacology,
Vanderbilt University School of Medicine, 23rd Ave. S at Pierce, Nashville,
TN 37232-6602. Tel.: 615-343-4495; Fax: 615-322-4707; E-mail: alan.brash@
vanderbilt.edu.
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
In the present study, we aimed to understand the mechanis- purification. The Aldrich Atmosbag used for conducting the
tic basis for the different preferences of LOX enzymes for LTA anaerobic incubations was purchased from Sigma-Aldrich.
Reaction Incubations and Extraction—The initial anaerobic
reactions on an analytical scale were conducted inside an
N2-inflated Aldrich Atmosbag. First, placed into the Aldrich
Atmosbag were an open vial containing the enzyme solution
(0.76 ␮M soybean LOX-1 in 1 ml of sodium phosphate, pH 7.5),
an open vial containing the substrate solution (20–880 ␮M 15S-
HPETE in 1 ml of sodium phosphate, pH 7.5), syringes, vial
caps, and a bottle of methanol. The Atmosbag was then sealed
and subjected to vacuuming and N2 inflation for three cycles.
Twenty minutes was given for the enzyme and substrate solu-
tions to reach equilibrium with the N2 atmosphere. Then the
reaction was started by mixing the enzyme and the substrate.
Reaction was stopped by the addition of an equal volume of
methanol, followed by extraction using an Oasis HLB cartridge
(Waters Corp.).
synthesis or double oxygenation. Some clues can be gleaned
from the literature. Rabbit reticulocyte 15-LOX or porcine leu-
kocyte 12-LOX (both being homologs of human 15-LOX-1),
when incubated with 15S-HPETE under anaerobic reactions,
gives a higher yield of 14,15-LTA4 at the expense of the double
oxygenation products (3, 7). Conversely, under higher than
normal oxygen pressures, potato 5-LOX can be forced to per-
form double oxygenation on 5S-HPETE (8). These studies
imply that LTA synthesis and double oxygenation are in com-
petition, and the concentration of molecular oxygen is a critical
determinant of which process dominates. The latter point can
be better understood when one considers that LTA synthesis
does not involve molecular oxygen, whereas the double oxygen-
ation reaction uses molecular oxygen as one of the two sub-
strates. Because the catalysis should be influenced by oxygen
concentrations in the enzyme active site, rather than in the
atmosphere per se, we hypothesized that the different prefer-
ences of LOX isoenzymes for LTA synthesis or double oxygen-
ation stem from different oxygen concentrations in their active
sites.
For the reactions in the presence of NDGA or 13S-HPODE,
80 ␮M NDGA or 13S-HPODE was included in the substrate
solution. For structural analysis of the products, the incuba-
tions were scaled up to a volume of 20 ml using otherwise the
same conditions. For incubations in 18O-enriched buffer, a vol-
ume of 0.5 ml was used, which is comprised of 0.4 ml of 96%
H218O and 0.1 ml of 500 mM sodium phosphate, pH 7.5.
Kinetic Measurements—For kinetic measurements, the anaer-
obic reaction of soybean LOX-1 (usually 0.2 ␮M unless noted
otherwise) with 15S-HPETE (50–500 ␮M) was monitored at
280 nm in an enclosed quartz cuvette using a PerkinElmer
Lambda-35 spectrophotometer. N2-saturated sodium phos-
phate buffer (pH 7.5), glucose oxidase, glucose, and catalase
were used to achieve the anaerobic conditions; subsequently,
HPLC analyses verified that no oxygenation products were
formed. The reaction volume is usually 1.1 ml. To avoid poten-
tial inhibition of alcohols on the enzyme activity, 15S-HPETE
originally dissolved in methanol was first added to the quartz
cuvette and then taken to dryness under a stream of N2. The
cuvette was then placed at the bottom of a plastic bag filled with
argon. 1 ml of N2-saturated buffer, 25 ␮l of glucose oxidase
(stock concentration: 827 Sigma-units/ml), 1 ␮l of catalase (the
stock solution was 5ϫ diluted from the commercial solution of
bovine liver catalase purchased from Sigma-Aldrich, C3155,
Ն35,000 units/mg protein), and 50 ␮l of glucose (2 M stock,
N2-saturated) was then added to the cuvette. The cuvette was
then capped, taken out of the bag, shaken for a few seconds, and
let standing for about 1 min. Reaction was started by the quick
addition of 6 ␮l of soybean LOX-1 stock solution (final concen-
tration 0.2 ␮M).
With this hypothesis, we reasoned that soybean LOX-1,
which catalyzes only double oxygenation with 15S-HPETE
under usual aerobic conditions, might show 14,15-LTA4 syn-
thase activity when molecular oxygen is excluded. In this paper,
we have described such an anaerobic reaction of soybean
LOX-1 with 15S-HPETE. As anticipated, we observed the for-
mation of the hydrolysis products of the putative 14,15-LTA4.
Moreover, as a mechanistically informative finding, we identi-
fied among the reaction products two novel 5,15-dihydroxy
compounds and their ␦-lactone derivatives. Based on their
chemical structures, conditions of formation, and evidence
from H218O incubations, we suggested that the 5,15-dihydroxy
compounds and their ␦-lactone derivatives result from nucleo-
philic attack by water (hydrolysis) and by the terminal carbox-
ylate anion (lactonization), respectively, of a novel cyclopropyl
epoxide formed via an LTA-synthase-related mechanism.
EXPERIMENTAL PROCEDURES
Materials—Soybean LOX-1 (soybean P1, purified) and nor-
dihydroguaiaretic acid (NDGA) were purchased from Cayman
Chemical (Ann arbor, MI). We determined that the purified
soybean LOX-1 employed in this work was ϳ40% lower in spe-
cific activity than advertised (possibly due to two cycles of
freeze-thawing), ϳ20 ␮mol/min/mg using the Axelrod assay
with sodium linoleate (9). Linoleic and arachidonic acids were
purchased from NuChek Prep Inc. (Elysian, MN). [1-14C]Ar-
achidonic acid was purchased from PerkinElmer Life Sciences.
15S-HPETE and 13S-HPODE were synthesized by reacting
soybean LOX-1 with arachidonic acid at pH 9 under the normal
aerobic conditions, followed by SP-HPLC purification (10).
5(RS)-HETE was chemically synthesized from arachidonic acid
(11), and the enantiomers were resolved by chiral column
HPLC as described (12). 5,15-diHETE was prepared similarly
HPLC Analysis and Purification—The extracted products
were analyzed by RP-HPLC and SP-HPLC using an Agilent
1100 HPLC system equipped with a diode array detector. The
products from [1-14C]15S-HPETE were analyzed simulta-
neously by the diode array detector and by a Flo-One A-100
radioactive detector (Radiomatic Instruments and Chemical
Co., Meriden, CT). The RP-HPLC analysis used a Waters Sym-
metry C18 column (5 ␮m, 0.46 ϫ 25 cm), an isocratic solvent
system of methanol/water/acetic acid (80:20:0.01 by volume),
using 5S-HETE and/or 5R-HETE as substrate for soybean and a flow rate of 1 ml/min. The SP-HPLC analysis used a Beck-
LOX-1, followed by reduction using NaBH4 and SP-HPLC man Ultrasphereꢀ silica column (5 ␮m, 0.46 ϫ 25 cm), an iso-
13428 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 18•APRIL 30, 2010

A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
cratic solvent system of hexane/isopropyl alcohol/acetic acid
(100:2:0.1 by volume for less polar products followed by a
strong solvent wash or 100:5:0.1 for polar products), and a flow
rate of 1 ml/min. Products 2, 3, 4, and 5 (see Fig. 1) were puri-
fied by SP-HPLC. Products 1, 6, and 7 were purified by SP-
HPLC followed by RP-HPLC. Separation of the two diaste-
reomers of product 4 was achieved using a Chiralpak AD-RH
column (5 ␮m, 0.46 ϫ 15 cm), an isocratic solvent system of
methanol/water/acetic acid (95:5:0.01 by volume), and a flow
rate of 0.5 ml/min. For analysis of product 5, we used the same
column, methanol/acetic acid (100:0.01 by volume), and a flow
rate of 1 ml/min.
Synthesis of HETEs and diHETE with trans,trans-Conjugated
Dienes—Thiyl radical-induced isomerization (mercaptoetha-
nol, UV light, room temperature (13, 14)) was used to convert
cis-trans- to trans,trans-conjugated dienes. Reaction condi-
tions had the starting material (2.5 mg, methyl ester of HETE or
diHETE) in 0.5 ml of isopropyl alcohol containing 0.5 ␮l of
mercaptoethanol in a quartz UV cuvette with Teflon cap. The
cuvette was submerged in a solution of aqueous 1 M NaCl that
served both to stabilize the temperature at Ͻ25 °C and absorb
UV radiation below 220 nm. A low pressure immersion UV
lamp rated with 3.5-watt output at 254 nm (part no. 12128-15,
Ace Glass Inc. (Vineland, NJ)) (caution: harmful UV radiation)
was placed about 15 cm above the cuvette. Aliquots of the reac-
tion were analyzed at 30-min intervals by argentation HPLC
using a cation exchange column (Sepralyte SCX 5-␮m, 25 ϫ
0.46 cm, originally purchased from Analytichem International
(later owned by Varian)) in the silver form (15, 16) with a sol-
vent of hexane/isopropyl alcohol/acetic acid in the proportions
70:30:0.1 (by volume) for mono-HETE methyl esters and 5,15-
diHETE methyl ester, a flow rate of 1 ml/min, and with UV
monitoring using an Agilent 1100 series diode array detector.
5-HETE and 15-HETE were isomerized in the conjugated
diene, leading to accumulation of the trans,trans-HETEs (with
two trans and two cis double bonds) with very minor peaks for
the more highly isomerized derivatives. 5,15-diHETE was
isomerized in one conjugated diene and, after 2–3 h of irradia-
tion in both, giving the all-trans derivative as the major product.
Derivatization and GC-MS Analysis—Catalytic hydrogena-
tions were performed in 100 ␮l of ethanol using about 1 mg of
palladium on alumina and bubbling with hydrogen for 2 min at
room temperature. The hydrogenated products were recovered
by the addition of water and extraction with ethyl acetate. TMS
ester TMS ether derivatives were prepared by treatment with
bis(trimethylsilyl)-trifluoracetamide (10 ␮l) and pyridine (2 ␮l)
at room temperature for 2 h. Subsequently, the reagents were
evaporated under a stream of nitrogen, and the samples were
dissolved in hexane for GC-MS.
FIGURE 1. RP-HPLC analysis of products from the anaerobic reaction of
soybean LOX-1 (0.38 ␮M) with 15S-HPETE (40 ␮M). The products were
eluted using a Waters Symmetry C18 column (0.46 ϫ 25 cm), a solvent system
of methanol/water/acetic acid (80:20:0.01 by volume), and a flow rate of 1
ml/min. Shown are the UV traces at 205, 235, and 270 nm monitored by an
Agilent diode array detector. The products are designated 1–7 based on the
order of elution.
ESI-LC-MS—ESI-LC-MS of purified product 4 and product
5 was performed using a Thermo Finnigan LC Quantum instru-
ment. A Waters Symmetry C18 column (0.2 ϫ 15 cm) was
eluted with acetonitrile/water/ammonium acetate (50 mM, 50
mM, 10 mM) at 0.2 ml/min. The heated capillary ion lens was
operated at 220 °C. Nitrogen was used as a nebulization and
desolvation gas. The electrospray potential was held at 4 kV.
Source-induced dissociation was set at Ϫ10 eV. Mass spectra
were acquired over the mass range m/z 100–500 at 2 s/scan
under the negative ion mode.
NMR—1H and 1H,1H COSY NMR spectra were recorded on
a Bruker DXR 600-MHz spectrometer at 298 K. The ppm values
are reported relative to residual non-deuterated solvent (␦ ϭ
7.16 ppm for C6H6).
RESULTS
An Anaerobic Reaction of Soybean LOX-1 with 15S-HPETE—
To test the hypothesis that soybean LOX-1 is capable of cata-
lyzing LTA synthesis in the absence of oxygen, initially we
incubated the enzyme (0.38 ␮M) with various concentrations of
15S-HPETE (20–440 ␮M) at pH 7.5 under N2 for 2 h. Although
analysis of reaction rates is complicated by the mixture of prod-
ucts formed (see below), subsequent analyses indicated that, for
example, 100 ␮M 15S-HPETE is fully consumed within 10 min.
Observed rates are presented in the supplemental material.
Reactions were stopped by the addition of an equal volume of
methanol, followed by extraction using an Oasis HLB cartridge.
RP-HPLC analysis revealed that the substrate 15S-HPETE, at
all concentrations tested, was completely converted to seven
new compounds, designated 1–7 based on the order of elution
(Fig. 1). We also examined the reaction carefully for the pro-
duction of fatty acid dimers because their formation would be
predicted if this reaction follows the same mechanism as the
well studied anaerobic reaction of soybean LOX-1 with linoleic
Analysis of the methyl ester trimethylsilyl ether derivatives of
the products was carried out by GC-MS in the positive ion
electron impact mode (70 eV), using a Thermo Finnigan Trace
DSQ ion trap instrument with the Xcalibur data system. Sam-
ples were injected at 150 °C, and after 1 min, the temperature
was programmed to 300 °C at 20 °C/min. Spectra collected dur-
ing elution of the GC peak (typically about 20 spectra) were
averaged for calculation of the isotopic compositions.
APRIL 30, 2010•VOLUME 285•NUMBER 18
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
HOOC
Product 1
O
O
HO
O
Product 6
Product 7
HOOC
HOOC
SCHEME 1. Structures of products 1, 6, and 7.
tetraenoic acid (15-KETE) (Scheme 1). Formation of the C15
aldehyde (product 1) and 15-KETE (product 7) is expected
because they are structural analogues of the major C13 aldehyde
and 13-KETE formed in the anaerobic reaction of the Fe(II)
enzyme with 13S-HPODE (18). Product 7 is a direct analogue of
threo-11-hydroxy-12,13S-trans-epoxyoctadec-9Z-enoic acid,
another known compound derived from 13S-HPODE by soy-
bean LOX-1 (22), and this threo-13-hydroxy-14,15-trans-ep-
oxyalcohol was originally identified by Corey and Mehrotra in
1983 (23).
FIGURE 2. RP-HPLC analysis of products from the anaerobic reaction of
soybean LOX-1 (0.38 ␮M) with 15S-HPETE (40 ␮M) in the presence of
NDGA (80 ␮M). As in Fig. 1, the products were eluted using a Waters Symme-
try C18 column (0.46 ϫ 25 cm), a solvent system of methanol/water/acetic
acid (80:20:0.01 by volume), and a flow rate of 1 ml/min. Shown are the UV
traces at 205, 235, and 270 nm monitored by an Agilent diode array detector.
Contribution of the Fe(III) Enzyme to the Anaerobic Reaction
with 15S-HPETE—To examine the products from the Fe(III)
enzyme, we took advantage of the fact that 13S-HPODE reacts
with Fe(II) soybean LOX-1 stoichiometrically to give Fe(III)
soybean LOX-1 (24). Thus, soybean LOX-1 (0.2 ␮M) was
reacted with [1-14C]15S-HPETE (40 ␮M) in the presence of 13S-
HPODE (80 ␮M). SP-HPLC with both UV and radioactivity
detection revealed that 2, 3, 4, and 5 were the main products
from the Fe(III) enzyme reaction (Fig. 3), the complementary
set of products compared with those formed from reactions of
the Fe(II) enzyme in the presence of NDGA or linoleic acid.
Identification of the Fe(III) Enzyme Products 2 and 3—Prod-
ucts 2 and 3 were identified as 8R,15S-dihydroxyeicosa-5Z,9E,
11E,13E-tetraenoic acid (8R,15S-Z,E,E,E-diHETE) and 8S,15S-
dihydroxyeicosa-5Z,9E,11E,13E-tetraenoic acid (8S,15S-Z,E,E,
E-diHETE) respectively, based on the same UV, mass spectra
(supplemental Figs. S3 and S4), and chromatographic proper-
ties they exhibited as the authentic standards. These 8,15-diols
with an all-trans-conjugated triene are characteristic hydrolysis
products of the unstable allylic epoxide, 14,15-LTA4.
acid and 13S-HPODE (17–19). However, SP-HPLC and RP-
HPLC analyses showed that this reaction gave no appreciable
amounts of C–C linked dimers and confirmed that products
1–7 constitute the main products.
Contribution of the Fe(II) Enzyme to the Anaerobic Reaction
with 15S-HPETE—Because soybean LOX-1 exists in two oxi-
dation states, Fe(II) and Fe(III), we next questioned which
enzyme form is responsible for the formation of the products.
NDGA, as a redox-based LOX inhibitor, inhibits the oxygenase
activity of LOX by reduction of Fe(III) LOX to Fe(II) LOX (20);
therefore, in the presence of NDGA, soybean LOX-1 would be
kept in the Fe(II) state, and only the products from the Fe(II)
enzyme would be observed.
The anaerobic reaction of soybean LOX-1 (0.38 ␮M) with
15S-HPETE (40 ␮M) was performed in the presence of NDGA
(80 ␮M) and subsequently analyzed by RP-HPLC. As shown in
Fig. 2, only products 1, 6, and 7 were formed in the presence of
NDGA, indicating that these are the products from the Fe(II)
enzyme. In addition, we noted that NDGA significantly accel-
erated the rate of reaction, especially at substrate concentra-
tions lower than 100 ␮M (e.g. see supplemental Fig. S1).
Under anaerobic conditions, linoleic acid also reduces Fe(III)
soybean LOX-1 to Fe(II) soybean LOX-1, with itself being con-
verted to a carbon-centered radical and then released from the
enzyme active site (21). Accordingly, linoleic acid should have
the same effect as NDGA does on the anaerobic reaction of
soybean LOX-1 with 15S-HPETE. Indeed, in the presence of
linoleic acid (150 ␮M), 15S-HPETE was converted to essentially
the same set of products as in the presence of NDGA (data not
shown).
Identification of the Fe(III) Enzyme Products 4 and 5—Prod-
ucts 4 and 5 display unusual UV spectra similar to yet distin-
guishable from that of the well known double oxygenation
product 5,15-dihydroxyeicosa-6E,8Z,11Z,13E-tetraenoic acid
(5,15-diHETE). In SP-HPLC solvents, the differences are the
more striking (cf. Fig. 4, A and B). LC-ESI-MS (negative ion
mode) showed that product 4 has the same relative molecular
mass, 336, as 5,15-diHETE (whereas under the same condi-
tions, product 5 was not detected; see below). GC-MS analysis
of the TMS ester TMS ether derivative of hydrogenated prod-
uct 4 established the 5,15-dihydroxy structure (supplemental Fig.
S5); the prominent ions at m/z 401 and 261 arise from ␣-cleav-
age at the 5-hydroxyl, and m/z 173 and 489 come from ␣-cleav-
age at the 15-hydroxyl. Thus, product 4 is a geometric isomer of
5,15-diHETE. Unlike the well known 5,15-diHETE containing
two cis-trans-conjugated diene chromophores (6), product 4
Identification of the Fe(II) Enzyme Products 1, 6, and 7—As
detailed in the supplemental material, products 1, 6, and 7 were
identified by UV, LC-MS, and by NMR. Product 1 is the C15
aldehyde 15-oxopentadeca-5Z,8Z,11Z,13E-tetraenoic acid,
product 6 is 13R-hydroxy-14S,15S-epoxyeicosa-5Z,8Z,11Z- contains four double bonds, all in the trans configuration as
trienoic acid, and product 7 is 15-oxoeicosa-5Z,8Z,11Z,13E- indicated by the coupling constants (J Ϸ 15 Hz) obtained from
13430 JOURNAL OF BIOLOGICAL CHEMISTRY
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
FIGURE 3. SP-HPLC analysis of products from the anaerobic reaction of
soybean LOX-1 (0.38 ␮M) with [1-14C]15S-HPETE (40 ␮M) in the presence
of 13S-HPODE (80 ␮M). A, the 14C radiochromatogram recorded by a radio-
active detector. B, the UV chromatogram at 235 and 270 nm recorded by a
diode array detector. The products were eluted using a Beckman Ultras-
phereꢁ silica column (0.46 ϫ 25 cm), a solvent system of hexane/isopropyl
alcohol/acetic acid (100:5:0.1 by volume), and a flow rate of 1 ml/min.
FIGURE 4. Comparison of the UV spectra of products 4 and 5 with that of
5,15-(E,Z,Z,E)-diHETE. A, the spectra in RP-HPLC solvent, methanol/water/
acetic acid (80:20:0.01 by volume). B, the spectra in SP-HPLC solvent, hexane/
isopropyl alcohol/acetic acid (100:5:0.1 by volume).
1H NMR (Fig. 5A and supplemental Table S2) and 1H,1H COSY
NMR analysis (supplemental Fig. S6).
To confirm the structure of product 4, we synthesized all-
trans-5,15-diHETE by thiyl radical-induced isomerization of
the cis double bonds in 5,15-diHETE (see “Experimental Proce-
dures”). The UV, chromatographic, and NMR properties of the
synthetic compound were identical to those of product 4. In
addition, comparison of the olefin regions of the NMR spectra
of synthetic 6E,8E-5-HETE and 11E,13E-15-HETE helped with
assignment of the individual olefin protons in all-trans-di-
HETE, in particular confirming that the H-6 signal is more
upfield of H-14 and thus allowing assignment of the rest of
the double bond proton signals from the COSY analysis
(supplemental Fig. S6).
acid (5,15-all-trans-diHETE), and product 5 is the correspond-
ing ␦-lactone.
Products 4 and 5 Are Each a Mixture of Diastereomers—Al-
though each appeared as a single peak on RP-HPLC and SP-
HPLC, each could be further resolved using a chiral HPLC col-
umn into a 50:50 mixture of two peaks with identical UV
spectra (supplemental Fig. S9). (Their NMR spectra are also
identical because there were no indications of a mixture of two
sets of signals in the spectra of product 4 or 5). Based on the
reasonable assumption that the original S configuration of the
substrate 15S-HPETE is retained in the 15-hydroxyl, we con-
clude that the peaks resolved on the chiral column are diaste-
reomers rather than enantiomers, epimeric at C-5. This was
confirmed using synthetic all-trans-diHETE prepared from a
2:1 mixture of 5S,15S- and 5R,15S-diHETE diastereomers. Sim-
ilar to product 4, this synthetic all-trans-diHETE chromato-
graphed as a single peak on RP- and SP-HPLC. On the Chiral-
pak AD-RH column, it resolved into the larger, earlier eluting
peak of 5S,15S followed by the later eluting 5R,15S diaste-
reomer. Mechanistic considerations that follow from the
H218O experiments further support the conclusion that prod-
Unlike product 4, product 5 had eluded detection by ESI-MS,
suggesting the lack of ionizable groups in the structure (e.g. as
esters, including lactones). This was confirmed by GC-MS anal-
ysis of the TMS ether derivative of hydrogenated product 5,
which gave a spectrum identical to that published for the TMS
ether derivative of hydrogenated ␦-lactone of 5,15-diHETE (25)
(supplemental Fig. S7). Diagnostic ions include m/z 99, 173, and
1
1
327. H NMR and H,1H COSY NMR analysis of product 5
determined the configurations of the four double bonds to be
all-trans (Fig. 5B and supplemental Fig. S8 and Table S2). Thus,
product 4 is 5,15-dihydroxyeicosa-6E,8E,11E,13E-tetraenoic ucts 4 and 5 are each a mixture of diastereomers.
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
from the 15-hydroperoxyl group in the substrate (Fig. 6A). Ions
containing the 5-hydroxyl were shifted by ϩ2 mass units (to
m/z 263, 313, and 491), whereas m/z 173, the characteristic
fragment ion of the 15-hydroxyl, remained unchanged.
By contrast, GC-MS analysis of the derivatized hydrogenated
product 5, the ␦-lactone of all-trans-5,15-diHETE, indicated no
18O incorporation in the molecule (Fig. 6B). (The mass spec-
trum is identical to that of product 5 from H216O incubations.)
The lack of 18O in the ␦-lactone ring clearly demonstrated that
product 5 was not formed by lactonization of product 4.
Instead, we surmise that the C-5 oxygen in the ␦-lactone ring of
product 5 must derive from the free carboxylate group in the
substrate.
A Cyclopropyl Epoxide as the Enzymatic Product—The for-
mation of products 4 and 5 can be explained if the true enzy-
matic product is an allylic 14,15-epoxide with a cyclopropane
ring (Fig. 7; see “Discussion”). This epoxide is opened up in one
of two ways. Either water reacts at the end of the conjugated
system to yield product 4 (with corresponding incorporation of
18O from H218O at C-5), or nucleophilic attack by the free car-
boxylate yields the ␦-lactone derivative with no involvement of
water in the transformation.
DISCUSSION
Background of This Study—Soybean LOX-1 was the first
LOX to be discovered and has since received the most extensive
mechanistic studies among the LOX enzymes (26, 27). Typi-
cally, the hallmarks of LOX catalysis, such as the antarafacial
relationship between hydrogen abstraction and oxygen inser-
tion, were first demonstrated for soybean LOX-1 and then reca-
pitulated in studies on other LOX enzymes. One notable excep-
tion, however, is the LTA synthase type activity. From a
historical point of view, because no such chemistry had been
described for soybean LOX-1, it was not immediately recog-
nized at the time of the discovery of LTA4 that the same
enzyme, 5-LOX, catalyzes the formation of 5S-HPETE and
LTA4. The recognition came a number of years later, when it
was demonstrated that both 5-LOX and LTA4 synthase activi-
ties involve stereoselective hydrogen abstraction as the initial
step, always co-elute during purification, and are inhibited to
similar extents by LOX inhibitors (28, 29).
FIGURE 5. 1H NMR analysis of products 4 (A) and 5 (B) in d6-benzene.
Shown is an expanded view of the region corresponding to olefinic pro-
tons. The splitting pattern of each olefinic proton is also illustrated. The
proton signals are assigned with aid of 1H,1H COSY NMR (supplemental
Figs. S6 and S8).
The Anaerobic Reaction in H218O—To understand the origin
of the hydroxyls in products 2, 3, 4, and 5, we conducted the
anaerobic reaction of soybean LOX-1 with 15S-HPETE in 18O-
enriched water (80% H218O), followed by HPLC purification of
the individual products, catalytic hydrogenation, TMS ether
TMS ester derivatization, and GC-MS analysis.
LTA Synthase Activity and LOX Dual Specificity—Both the
primary LOX activity and the secondary LTA synthase activity
start with hydrogen abstraction, yet at two different positions.
Therefore, it has been suggested that LTA synthase activity is a
consequence of dual specificity (i.e. the ability of the enzyme to
abstract hydrogens from two different positions and thus make
two distinct hydroperoxides) (29). Consistent with this sugges-
tion, leukocyte 5-LOX gives the 8S-hydroperoxide from
8,11,14-eicosatrienoic acid (compared with the 5S-hydroper-
oxide from arachidonic acid) (30, 31). Similarly, mammalian
15-LOX-1 usually gives a mixture of 15S-HPETE and 12S-
HPETE from arachidonic acid (32). However, dual specificity
alone does not ensure LTA synthase type activity; for instance,
the dual specificity of soybean LOX-1 is evidenced by the for-
In the mass spectrum of the derivatized pair of 8,15-diols
with all-trans-conjugated trienes (products 2 and 3) from the
H218O reaction, the ions containing the 8-hydroxyl were all
shifted by ϩ2 mass units (i.e. ions at m/z 303 to 305, 359–361,
and 489–491), whereas the m/z 173 fragment containing the
15-hydroxyl remained unchanged (supplemental Figs. S10
and S11). Quantitative analysis of the isotopic ratios revealed
that the 8-hydroxyl derives exclusively from water, whereas the
15-hydroxyl derives from the 15-hydroperoxyl of the substrate.
In accord with the results on the 8,15-diols and in sharp con-
trast to the known mechanisms of formation of 5,15-diHETE
via double oxygenation, GC-MS analysis of derivatized hydro-
genated all-trans-5,15-diHETE (product 4), established that mation of 8S,15S-diHPETE and 5S,15S-diHPETE in its double
the 5-hydroxyl comes from water and the 15-hydroxyl comes oxygenation reaction with 15S-HPETE (6), yet no LTA syn-
13432 JOURNAL OF BIOLOGICAL CHEMISTRY
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
FIGURE 6. GC-MS (electron ionization) analysis of TMS-derivatized hydrogenated products 4 and 5 from the anaerobic reaction in H218O. A, the mass
spectrum of the TMS ether TMS ester derivative of hydrogenated product 4. B, the mass spectrum of the TMS ether derivative of hydrogenated product 5.
thase type activity has ever been detected for this enzyme under without interference. By contrast, at high active site oxygen
the usual aerobic conditions.
concentrations, as in soybean LOX-1, double oxygenation
LTA Synthase Activity and Active Site Oxygen—We hypoth- occurs so fast that LTA synthase type activity will be fully sup-
esize that oxygen concentration in the enzyme active site is an pressed. To relieve this potential suppression of LTA synthase
additional factor that determines whether LTA synthase type type activity imposed by molecular oxygen, here we performed
activity will be present in a LOX or not, through influence on the reaction of soybean LOX-1 with 15S-HPETE under anaer-
the rate of the competing process, double oxygenation. When obic conditions. Consistent with our hypothesis, we observed
oxygen concentration is low, as we would propose in the 5-LOX under these conditions the formation of 8(RS),15S-Z,E,E,E-di-
active site, double oxygenation barely occurs due to deprivation HETEs, the expected hydrolysis products from 14,15-LTA4 (or
of oxygen co-substrate, so LTA synthase activity will occur the cis-epoxide isomer; see below). This result strongly sup-
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
acid autoxidation (36) and in hemo-
globin-catalyzed transformation of
fatty acid hydroperoxides (37, 38), is
also feasible on purely chemical
grounds (supplemental Fig. S12). The
key difference of the two pathways is
the order of the two chemical steps,
hydrogen abstraction and peroxide
bond cleavage; whereas the Fe(III)
LOX pathway involves hydrogen
abstraction followed by peroxide
bond cleavage, the Fe(II) LOX path-
way employs the same two steps in
the reverse order. To clarify this
issue in the present case, we dis-
sected the anaerobic reaction of
soybean LOX-1 with 15S-HPETE
into two components: one catalyzed
by the Fe(II) enzyme and the other
by the Fe(III) enzyme, using NDGA
and 13S-HPODE to keep the
enzyme ferrous and ferric, respec-
tively. We found that 8(RS),15S-
Z,E,E,E-diHETEs are formed in the
presence of 13S-HPODE but not in
the presence of NDGA, thus deter-
mining unambiguously that the
Fe(III) enzyme is the catalytic spe-
15S-HPETE
H
H
Fe(III) - OH
HOOC
OOH
H-7 atom abstraction
Fe(II) - H2O
HOOC
HOOC
OOH
O
Fe(III) - OH
HOOC
O
HOOC
O
intramolecular δ−lactonization
H2O
HOOC
HO
OH
O
OH
O
product 4
product 5
FIGURE 7. The proposed mechanism of formation of products 4 and 5.
ports our view that the soybean LOX-1 has all of the essential cies in the soybean LOX-1-catalyzed formation of 14,15-LTA4
elements to fulfill the role of a LTA synthase, yet this potential and the corresponding 8,15-diol hydrolysis products. Our
has been obscured by the presence of high concentrations of result agrees well with the early studies using stereospecifically
oxygen in the active site, which favors the double oxygenation tritium-labeled arachidonic acid that established hydrogen
reaction.
abstraction as the first irreversible step in LT synthesis (28) and
An oxygen channel in the LOX protein might normally target also agrees with the studies using LOX inhibitors that demon-
O2 to the correct position on the reacting substrate (33, 34), or strated the inhibitory effect of NDGA on LT formation from
alternatively there could be an appropriately positioned oxygen HPETEs, although at these early times the mechanism of inhi-
pocket in the active site (35). Either way, oxygen could be well bition of NDGA (3, 29) (i.e. by reduction of Fe(III) LOX to Fe(II)
positioned for one substrate (e.g. arachidonic acid in mamma- LOX) had yet to be clarified (20).
lian 5-LOX) and out of place for another (5S-HPETE in 5-LOX)
The Proposed Cyclopropyl Epoxide Intermediate Leading to
while adequately positioned for either arachidonic acid or 15S- 5(RS),15S-All-trans-diHETEs and the ␦-Lactone Derivatives—
HPETE in soybean LOX. It is expected that the Km(O2) for The most intriguing finding in the present study is probably the
soybean LOX-1 using 15S-HPETE as substrate would be con- detection of the novel compounds, 5(RS),15S-all-trans-di-
siderably lower than the Km(O2) for mammalian 15-LOX using HETEs and the ␦-lactone derivatives. Inspired by the structural
the same substrate or the Km(O2) for 5-LOX using 5S-HPETE. similarities of 5(RS),15S-all-trans-diHETEs to 8(RS),15S-
These values for fatty acid hydroperoxide substrates remain to Z,E,E,E-diHETEs, the shared involvement of the Fe(III) enzyme
be determined. However, evidence does exist that suggests a in the formation of both, and similar incorporations of H218O,
high Km(O2) for LOX that normally exhibit LTA synthase activ- we propose that the 5(RS),15S-all-trans-diHETEs are also
ity; e.g. a large Km(O2) can be deduced from the observation that hydrolysis products of an unstable intermediate, formed in a
potato 5-LOX shows double oxygenation activity with mechanism with parallels to the synthesis of 14,15-LTA4. A
5S-HPETE only under high oxygen pressures (8). It would be of feature of our proposal is involvement of first the ferric
interest to test whether a similar switch occurs with mamma- enzyme and then the ferrous species in activation at the two
lian 5-LOX or 15-LOX under hyperbaric conditions.
pentadiene units, followed by intramolecular coupling of
The Oxidation State of the Enzyme Involved in LTA Synthesis— two radicals to give an unstable epoxide, which, upon
Next, we questioned which enzyme form, Fe(III) or Fe(II) soy- hydrolysis, gives the 5,15-diols containing four trans double
bean LOX-1, is responsible for the formation of 8(RS),15S- bonds. Notably, a directly analogous mechanism can explain
Z,E,E,E-diHETEs. Although it is widely accepted that the LTA4 or 14,15-LTA4 synthesis.
Fe(III) LOX accounts for LTA synthesis, an alternative Fe(II)
As illustrated in Fig. 7, similar to 14,15-LTA4 formation,
LOX pathway, originally proposed for LT formation in fatty hydrogen abstraction by the Fe(III) enzyme is the initial step,
13434 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 18•APRIL 30, 2010

A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
but it occurs at C-7 rather than C-10 to form a carbon-centered
radical delocalized over the C-5–C-9 pentadiene. This initial
step produces the Fe(II) enzyme, which then induces homolytic
cleavage of the O–O bond of the 15-hydroperoxide group to
give an alkoxyl radical at the C-11–C-15 pentadiene. At this
stage, the reacting intermediate exists as an oxygen-centered
and carbon-centered biradical. Due to its intrinsic instability,
this biradical would rearrange, by reacting the oxygen-centered
radical with the nearby double bonds to form an epoxide group
and a second carbon- centered radical, followed by combina-
tion of the two carbon-centered radicals to form a new C–C
bond, to give a covalently complete compound, an allylic
14,15-epoxide with a cyclopropane ring composed of C-9,
C-10, and C-11, namely 14,15-epoxy-[9,10,11-cyclopropyl]-
eicosa-5Z,7E,13E-trienoic acid.
Similar to other allylic epoxides, at non-alkaline pH values,
this cyclopropyl epoxide may react with even the weakest
nucleophiles, such as water in aqueous solutions, to give
5(RS),15S-all trans-diHETEs or with the carboxylate group in
the molecule itself to give the corresponding ␦-lactones.
Because water is involved in the former but not the latter proc-
ess, only the 5(RS),15S-diols and not the ␦-lactones incorporate
water at C-5, exactly what we found using H218O. Finally, as a
subtle point of the proposed mechanism, the cyclopropane ring
is formed only in the covalently complete final product, as
opposed to in any radical intermediates, because such radical
intermediates containing a cyclopropane ring and an adjacent
radical would be highly energetically unfavorable (39).
FIGURE 8. Comparison of the aerobic and anaerobic reactions of soybean
LOX-1 with 15S-HPETE. Aerobic reaction is initiated by the H-7 and H-10
hydrogen abstractions (pro-R hydrogens, designated as in arachidonic acid
rather than 15S-HPETE) with 15S-HPETE in the reversed orientation, antarafa-
cial oxygenation giving the 5S,15S-diHPETE and 8S,15S-diHPETE products,
respectively (6, 44, 45). Anerobically, analogous hydrogen abstractions can
account for formation of the cyclopropyl epoxide and 14,15-LTA4, respec-
tively, followed by non-enzymatic hydrolysis to give the stable diol end
products.
The stereochemistry of the epoxide group in the putative
14,15-LTA4 and the cyclopropyl epoxide intermediates cannot
be determined based on the analysis of the hydrolysis products
in the present study. Although perhaps not widely recognized,
LOX-catalyzed formation of the cis-epoxy isomer of allylic lipoxygenases. An example from the present study is formation
epoxides has been documented, and the cis-epoxy isomer by the Fe(II) enzyme of the threo-13-hydroxy-14,15-trans-ep-
would hydrolyze to the same set of products as the trans-ep- oxyalcohol, the 15-ketone, and C15 aldehyde. These reactions
oxide isomer, with only a subtle difference in the product ratios strongly resemble those of two unusual lipoxygenases that
(8).
appear to be structurally intact and yet catalyze no detectable
Conclusions—In summary, our study demonstrates the com- formation of fatty acid hydroperoxides with any natural poly-
petition between dioxygenation, which requires the presence of unsaturated fatty acid. One of these, a specific LOX enzyme in
appropriately positioned molecular oxygen in the LOX active maize (ZmLOX6) acts as a hydroperoxide lyase (40), forming
site, and leukotriene biosynthesis, which proceeds anaerobi- aldehydes of the type found with the anaerobic soybean LOX-1.
cally. For an enzyme such as soybean LOX-1 in which O2 is The other is the epidermal LOX-3 of mammalian skin, known
readily available for dioxygenation reactions, leukotriene bio- from genetic studies to be required for proper differentiation in
synthesis is not observed until the enzyme is placed in an anaer- the epidermis and which displays hydroperoxide isomerase
obic environment. Conversely, we surmise that in LOX activity (41, 42), forming epoxyalcohols and ketones, again sim-
enzymes in which leukotriene synthesis occurs under aerobic ilarly to the anaerobic soybean LOX-1. In both cases, a lack of
conditions, molecular oxygen is less readily available in the available oxygen within the active site may contribute to both
active site. In the case of soybean LOX-1, the dioxygenase reac- the lack of activity with natural fatty acids and the acquisition of
tions on 15S-HPETE are initiated via hydrogen abstractions at new activities with fatty acid hydroperoxides. The concept of a
C-7 and C-10, giving 5S,15S-diHETE and 8S,15S-diHETE limited access to oxygen within the lipoxygenase active site as a
respectively, and the LTA synthase type reactions are initiated basis for new activities might also be extended to include the
in the same way under anaerobic conditions (Fig. 8). The hydro- plant “type-II” LOX enzymes, such as soybean LOX-3, that cat-
gen abstraction at C-7 leads to formation of the novel cyclopro- alyze mainly nonspecific peroxidation of fatty acids and their
pyl-containing epoxide, which in turn gives the 5,15-diols with esters and further conversion of the hydroperoxide products to
all-trans double bonds and the corresponding ␦-lactones.
ketones (9, 43). In their case, it appears that hydrogen abstrac-
In addition to driving the reaction initiated by the Fe(III) tion is followed by release of the fatty acid radical and that
enzyme down the LT synthase pathway, a lack of active site reaction with oxygen occurs outside the LOX active site. The
oxygen may play a role in other non-canonical reactions of enzyme, being left in the ferrous state, would in turn react with
APRIL 30, 2010•VOLUME 285•NUMBER 18
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A Novel Arachidonic Acid Epoxide with Cyclopropyl Ring
327–332
fatty acid hydroperoxides to give alkoxyl radicals that lead even-
19. Garssen, G. J., Vliegenthart, J. F., and Boldingh, J. (1972) Biochem. J. 130,
435–442
tually to ketones. A lack of access to oxygen within the active
site may prompt this outcome.
20. Kemal, C., Louis-Flamberg, P., Krupinski-Olsen, R., and Shorter, A. L.
(1987) Biochemistry 26, 7064–7072
Finally, we note the implications for the mechanism of leuk-
otriene A4 synthesis based on the proposed mechanism of for-
mation of the epoxy-cyclopropyl fatty acid. The involvement of
21. de Groot, J. J., Garssen, G. J., Vliegenthart, J. F., and Boldingh, J. (1973)
Biochim. Biophys. Acta 326, 279–284
both the ferric and ferrous iron and the production of a fleeting 22. Garssen, G. J., Veldink, G. A., Vliegenthart, J. F., and Boldingh, J. (1976)
Eur. J. Biochem. 62, 33–36
biradical species in both types of reaction are strongly implied.
23. Corey, E. J., and Mehrotra, M. M. (1983) Tetrahedron Lett. 24, 4921–4922
24. de Groot, J. J., Garssen, G. J., Veldink, G. A., Vliegenthart, J. F., and Bold-
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Acknowledgments—We thank Harold W. Gardner and Ned. A. Porter
for valuable discussions and William E. Boeglin for chemical synthesis
of all-trans-5,15-diHETE and trans,trans-HETEs.
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13436 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 18•APRIL 30, 2010

Enzymology:
Formation of a Cyclopropyl Epoxide via a
Leukotriene A Synthase-related Pathway in
an Anaerobic Reaction of Soybean
Lipoxygenase-1 with 15S
-Hydroperoxyeicosatetraenoic Acid:
EVIDENCE THAT OXYGEN ACCESS IS
A DETERMINANT OF SECONDARY
REACTIONS WITH FATTY ACID
HYDROPEROXIDES
Yuxiang Zheng and Alan R. Brash
J. Biol. Chem. 2010, 285:13427-13436.
doi: 10.1074/jbc.M109.084632 originally published online March 1, 2010
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