Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: Substrate chirality as a determinant of the cis or trans epoxide confi guration

Article abstract of DOI:10.1194/jlr.M033746

Leukotriene (LT)A 4 and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA 4 is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide confi guration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities suffi cient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15 S-hydroperoxy-eicosatetraenoic acid (15 S-HPETE) to the LTA analog 14 S ,15 S-trans-epoxy-eicosa-5 Z ,8 Z ,10 E ,12 E-tetraenoate, confi rming the proposed structure of eoxin A 4 . Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5 S-HPETE to the trans epoxide LTA 4 and converts 5 R-HPETE to the cis epoxide 5-epi-LTA 4 , establishing substrate chirality as a determinant of the cis or trans epoxide confi guration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.Copyright

Full text of DOI:10.1194/jlr.M033746

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Biosynthesis, isolation, and NMR analysis of leukotriene A
epoxides: substrate chirality as a determinant of the
cis or trans epoxide configuration
Jing Jin, Yuxiang Zheng,1 William E. Boeglin, and Alan R. Brash2
Department of Pharmacology and the Vanderbilt Institute of Chemical Biology, Vanderbilt University,
Nashville, TN 37232
Abstract Leukotriene (LT)A4 and closely related allylic
epoxides are pivotal intermediates in lipoxygenase (LOX)
pathways to bioactive lipid mediators that include the leu-
kotrienes, lipoxins, eoxins, resolvins, and protectins. Al-
though the structure and stereochemistry of the 5-LOX
product LTA4 is established through comparison to synthetic
standards, this is the exception, and none of these highly
unstable epoxides has been analyzed in detail from enzymatic
synthesis. Understanding of the mechanistic basis of the cis or
trans epoxide configuration is also limited. To address these
issues, we developed methods involving biphasic reaction
conditions for the LOX-catalyzed synthesis of LTA epoxides
in quantities sufficient for NMR analysis. As proof of con-
cept, human 15-LOX-1 was shown to convert 15S-hydroper-
oxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog
14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, con-
firming the proposed structure of eoxin A4. Using this method-
ology we then showed that recombinant Arabidopsis AtLOX1,
an arachidonate 5-LOX, converts 5S-HPETE to the trans ep-
oxide LTA4 and converts 5R-HPETE to the cis epoxide 5-epi-
LTA4, establishing substrate chirality as a determinant of the
The biosynthetic pathway, unraveled three decades ago,
is a model for related transformations to anti-inflamma-
tory mediators including the lipoxins, eoxins, resolvins,
protectins, and maresins (2–5). 5-Lipoxygenase (5-LOX)
catalyzes the first two steps in leukotriene biosynthesis,
namely the stereospecific oxygenation of arachidonic acid
to 5S-hydroperoxy-eicosatetraenoic acid (5S-HPETE),
and dehydration of the hydroperoxide to LTA4, the allylic
5,6-trans-epoxide from which the bioactive leukotrienes
are derived (Fig. 1) (6–9). The importance of this pivotal
intermediate is reflected in the extensive efforts directed
toward perfecting the total synthesis of LTA4 and of closely
related 5,6-epoxide isomers (as reviewed in Refs. 10–12).
LTA4 is highly unstable in physiological buffer at pH
7.4, with a half-life estimated as approximately 3 s at 25°C
and 18 s at 4°C (13). Despite this instability, LTA4 was de-
tected as an evanescent intermediate in short-term incuba-
tions of arachidonic acid with human leukocytes (6), and
the radiolabeled product was isolated and, as the methyl
ester derivative, shown to have similar chromatographic
mobility and hydrolysis under acidic conditions as a syn-
thetic standard (14). The stereochemistry has heretofore
not been established directly on the biosynthetic product
but was deduced from an understanding of the biosyn-
thetic pathway and by study of the reactions of synthetic
LTA4 and related isomers in transformations to the stable,
bioactive leukotrienes LTB4 and LTC4 (15–17). Herein we
report the isolation and purification of LTA epoxides as
products of LOX enzymes in quantities sufficient for NMR
analysis of the stereoconfigurations. The isolation method
cis or trans epoxide configuration.
The results are recon-
ciled with a mechanism based on a dual role of the LOX
nonheme iron in LTA epoxide biosynthesis, providing a ra-
tional basis for understanding the stereochemistry of LTA
epoxide intermediates in LOX-catalyzed transformations.—
Jin, J., Y. Zheng, W. E. Boeglin, and A. R. Brash. Biosynthe-
sis, isolation, and NMR analysis of leukotriene A epoxides:
substrate chirality as a determinant of the cis or trans epox-
ide configuration. J. Lipid Res. 2013. 54: 754–761.
Supplementary key words 5-lipoxygenase • 15-lipoxygenase • hy-
droperoxy-eicosatetraenoic acid • nuclear magnetic resonance
The leukotrienes (LTs) are a family of lipid mediators
derived from arachidonic acid and implicated in the
pathogenesis of asthma and other inflammatory diseases (1).
Abbreviations: HPETE, hydro(pero)xyeicosatetraenoic acidLT,
leukotriene; LOX, lipoxygenase; 5-oxo-ETE, 5-oxo-eicosatetraenoic acid;
RP-HPLC, reverse phase high-pressure liquid chromatography; SP-HPLC,
straight phase high-pressure liquid chromatography; UV, ultraviolet.
1 Present address: Department of Systems Biology, Harvard Univer-
sity, Boston, MA 02115
This work was supported by National Institutes of Health grant GM-015431.
Its contents are solely the responsibility of the authors and do not necessarily
represent the official views of the National Institutes of Health or other granting
agencies.
2 To whom correspondence should be addressed.
e-mail: alan.brash@vanderbilt.edu
Manuscript received 29 October 2012 and in revised form 12 December 2012
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of one figure and supplemen-
tary data.
Published, JLR Papers in Press, December 13, 2012
DOI 10.1194/jlr.M033746
Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.
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solvent system of hexane/isopropanol (100/1, by volume) run at
a flow rate of 4 ml/min (Beckman) . The enantiomeric HPETE
methyl esters were resolved using a semi-preparative, 25 × 1 cm
Chiralpak AD column (Chiral Technologies Inc., West Chester,
PA) with a solvent system of hexane/methanol (100:2, by vol-
ume) run at a flow rate of 4 ml/min (21).
To prepare the corresponding free acids, the purified HPETE
methyl esters (1 mg) in 2 ml methanol/methylene chloride
(10:1, by volume) were brought to room temperature, and an
equal volume of 1 M KOH was added. The sample was mixed and
kept at room temperature under an atmosphere of argon with
occasional sonication in a water bath. After 20 min, the sample
was acidified to pH 4.5 and extracted with an equal volume of
methylene chloride. The organic phase was washed twice with
water and then evaporated to dryness under a stream of nitrogen.
The dried sample was redissolved in methanol and stored at
Ϫ30°C. HPETEs were quantified based on the conjugated diene
chromophore at 236/237 nm, ␧ = 25,000 (10 ␮g/ml = 0.75 AU).
Expression and purification of human 15-LOX-1 and
Arabidopsis thaliana AtLOX1
The cDNA of human 15-LOX-1 was subcloned into the pET3a
vector (with an N-terminal His6 tag), and the protein was ex-
pressed in BL21 cells. A typical preparation of a 100-ml culture
was carried out as follows: 100 ml of 2XYT medium containing
100 ␮g/ml ampicillin was inoculated with a single colony of
h15-LOX-1-His in BL21 cells and grown at 37°C at 250 rpm
until OD600 reached 0.8. Isopropyl ␤-D-1-thiogalactopyranoside
(0.5 mM) was then added to the culture, which was grown at
16°C, 220 rpm for 4 days. The cells were spun at 5,000 g for
20 min in a Beckman Avanti J-25I centrifuge, washed with 40 ml
of 50 mM Tris (pH 7.9), pelleted again at 5,000 g for 20 min, and
resuspended in 10 ml of 50 mM Tris (pH 8.0), 500 mM NaCl,
20% glycerol, and 100 ␮M PMSF. The spheroplasts were soni-
cated five times for 10 s using a model 50 Sonic Dismembrator
(Fisher Scientific, Pittsburgh, PA) at a setting of 5. CHAPS deter-
gent was added at a final concentration of 1% (w/w), and the
sample was kept on ice for 20 min. The resulting membranes
were spun at 5,000 g for 20 min at 4°C. h15-LOX-1 activity was
present in the supernatant. The supernatant was loaded on a
nickel-NTA column (0.5 ml bed volume; Qiagen, Gaithersburg,
MD) equilibrated with 50 mM Tris buffer (pH 8.0), 500 mM
NaCl. The column was then washed with the equilibration, buffer
and the nonspecific bound proteins were eluted with 50 mM Tris
buffer (pH 8.0), 500 mM NaCl, and 50 mM imidazole. The h15-
LOX-1 was then eluted with 50 mM Tris buffer (pH 8.0), 500 mM
NaCl, and 250 mM imidazole. Fractions of 0.5 ml were collected
and assayed for the LOX activity. The positive fractions were dia-
lyzed against 50 mM Tris buffer (pH 7.5) and 150 mM NaCl. The
purity of the enzyme preparations was determined by SDS-PAGE
and Coomassie Blue staining; the prominent band of h15-LOX-1
accounted for about 80% of the total protein.
Fig. 1. The 5-LOX catalyzed pathway of LTA4 biosynthesis. 5-LOX
oxygenates arachidonic acid to 5S-HPETE and then dehydrates the
hydroperoxide to LTA4. Shown in gray, metabolism by LTA4 hydro-
lase produces the bioactive leukotriene LTB4.
we developed here was successfully applied earlier to al-
lene oxides (18) and to analysis of an LTA-related epoxide
synthesized by a catalase-related hemoprotein (19), al-
though not heretofore applied to lipoxygenase reactions.
We identify substrate chirality as a determinant of the cis
or trans configuration of the LTA epoxide and propose a
model that predicts how these products are derived in the
enzymatic transformation.
MATERIALS AND METHODS
Materials
Arachidonic acid and its methyl ester were purchased from
NuChek Prep Inc. (Elysian, MN). Soybean LOX-1 (lipoxidase,
type V) and ␣-tocopherol were purchased from Sigma (St. Louis,
MO). 15S-HPETE was synthesized by reacting soybean LOX-1
with arachidonic acid followed by straight-phase HPLC (SP-
HPLC) purification.
Synthesis and purification of enantiomeric HPETEs
Racemic hydroperoxides were prepared by vitamin E-con-
trolled autoxidation (20). Arachidonic acid methyl ester (500 mg)
was transferred to a 2 liter round-bottomed flask, mixed with
10% (w/w) ␣-tocopherol, and evaporated to dryness. The flask
filled with oxygen, capped, and placed in an oven at 37°C. The
oxygen was replenished daily. After 3 days, the lipid was dissolved
in 10 ml of methylene chloride and stored at Ϫ30°C. The autoxi-
dation sample was fractionated and partly purified using a 5 g
silica Bond-Elut (Varian, Palo Alto, CA) with the solvents of
hexane/ethyl acetate (three fractions of 10 ml of 95:5, followed
by three of 10 ml of 90:10 and three of 10 ml of 85:5, v/v); the
HPETE methyl esters were eluted in fractions 6 through 9. Race-
mic 5-HPETE and 15-HPETE methyl esters were then separated
from other positional HPETE isomers by SP-HPLC using a semi-
preparative Ultrasphere 10 ␮m silica column (25 × 1 cm) with a
The cDNA of Arabidopsis thaliana AtLOX1 was subcloned into
the pET14b vector (with an N-terminal His6 tag). The protein
was expressed in Escherichia coli BL21 (DE3) cells and purified by
nickel affinity chromatography according to a previously pub-
lished protocol (22).
Biphasic reaction conditions for preparation of
LTA epoxides
Enzyme reactions were performed at 0°C, with the HPETE
substrate initially in hexane (5 ml, bubbled for 30 min before use
with argon to decrease the O2 concentration, and containing
ف200 ␮M HPETE) layered over the recombinant LOX enzyme
(1–2 mg; ف20 nmol) in 400 ␮l of Tris buffer (pH 7.5 for h15-
LOX-1 and pH 6.0 for AtLOX1). The reaction was initiated by
Structural analysis of leukotriene A epoxides
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vigorous vortex mixing of the two phases. After 1.5 min, the
the hexane phase before and after vortex mixing provided
quick feedback on the extent of reaction. In the example
shown in Fig. 2, a new conjugated triene chromophore
hexane phase was collected and scanned from 200 to 350 nm in
ultraviolet (UV) light by using a Perkin-Elmer Lambda-35 spec-
trophotometer. The hexane phase was evaporated to about 2 ml
under a stream of nitrogen, treated with ethanol (20 ␮l) and
ethereal diazomethane for 10 s at 0°C, rapidly blown to dryness,
and kept in hexane at Ϫ80°C until further analysis.
typical of an LTA-type epoxide (␭
280 nm) is evident
max
after mixing. The substrate concentration has diminished
markedly and is only partially replaced by the conjugated
triene. Hydrolysis of the epoxide produces 8,15- and 14,15-
dihydroxy derivatives that do not extract into hexane,
especially at the elevated pH of 7.5, and the same applies
to any dioxygenation products or epoxyalcohol derivatives
(23). Analysis of the aqueous phase from these biphasic
reactions revealed the presence of dihydro(pero)xy deriv-
atives (prominently with all-trans conjugated trienes, ␭max
269 nm, typical of epoxide hydrolysis products, but includ-
ing dihydroperoxides) as well as a mixture of more polar
derivatives with trans-trans conjugated dienes (not identi-
fied, but possibly epoxyalcohols formed from conjugated
triene-containing dihydroperoxides). Overall, about 60%
of the starting 15S-HPETE was consumed, with an estimated
recovery of LTA-type epoxide under the above reaction
conditions of 10–15% (taking into account the molar
absorbance of the conjugated trienes is about 1.5–2 times
the value for a conjugated diene) (24, 25), with the balance
of products being accounted for mainly by the nonextract-
able, more polar derivatives.
After preparing the methyl esters of the hexane extract
by brief reaction with diazomethane at 0°C, the remaining
15S-HPETE and its products were analyzed on RP-HPLC
and SP-HPLC using conditions suitable for LTA-type ep-
oxides (13, 26). Fig. 3 illustrates RP-HPLC analysis with
UV detection at 270 nm. The unreacted 15S-HPETE (at
7.2 min retention time, the largest peak on the chromato-
gram but weakly detected at 270 nm) is immediately fol-
lowed by a minor keto derivative (at 7.6 min retention
time, a conjugated dienone, ␭max 281 nm) and then by a well-
resolved peak of the putative LTA-type epoxide (at 9.7 min,
HPLC analyses
Aliquots of the methylated hexane phase were analyzed by RP-
HPLC using a Waters Symmetry column (25 × 0.46 cm) using a
solvent of MeOH/20 mM triethylamine (pH 8.0) (90/10 by vol-
ume) at a flow rate of 1 ml/min, with on-line UV detection (1100
series diode array detector; Agilent) (19). Further purification
was achieved by SP-HPLC using a Beckmann Ultrasphere 5 ␮m
silica column (25 × 0.46 cm) using a solvent of hexane/trieth-
ylamine (100/0.5) run at 1 ml/min.
NMR analysis
1
1H NMR and H,1H COSY NMR spectra were recorded on a
Bruker AV-III 600 MHz spectrometer at 283 K. The parts/million
values are reported relative to residual nondeuterated solvent (␦ =
7.16 ppm for C6H6). Typically, 1,024 scans were acquired for a
1-D spectrum on ف20 ␮g of LTA epoxide.
GC-MS analysis of 5-oxo-ETE
The methoxime derivative was prepared by treatment with
methoxylamine hydrochloride (10 mg/ml in pyridine) at room
temperature for 16 h. The trimethylsilyl ester derivative was then
prepared using bis(trimethylsilyl)-trifluoroacetamide/pyridine
(12 ␮l, 5:1, v/v) for 2 h at room temperature. Analysis of the
trimethylsilyl ester methoxime derivative was carried out in the
positive ion electron impact mode (70 eV) using a Thermo Finni-
gan Trace DSQ ion trap GC-MS with the Xcalibur data system.
Samples were injected at 150°C, and after 1 min the temperature
was programmed to 300°C at 20°C/min. The spectrum was re-
corded by repetitive scanning over the range of 50–500 m/z.
a conjugated triene, ␭
280 nm). This product was col-
max
lected from SP-HPLC runs with a solvent of hexane con-
taining 0.1% triethylamine. The pooled aliquots of LTA
epoxide methyl ester (ف20 ␮g) were analyzed by 1H-NMR
in d6-benzene (Fig. 4). The 2-D COSY spectrum (described
RESULTS
Method development, preparation, and analysis of
14,15-LTA4
To prepare and isolate LTA epoxides, we used a bipha-
sic reaction system, kept at 0°C, with the HPETE substrate
dissolved in hexane and a highly active LOX enzyme in
aqueous buffer. Upon vigorous mixing, the more polar hy-
droperoxide partitions into the aqueous phase and reacts
with enzyme, and the less polar epoxide product instantly
back-extracts into the hexane and is thereby protected
from hydrolysis. For the initial development and valida-
tion of the method, we required an enzyme capable of
LTA-type epoxide synthesis and with the highest possible
catalytic activity. Our preparation of recombinant human
15-LOX-1 proved suitable; it converts its primary product,
15S-HPETE, to several derivatives, including the LTA4 ana-
log 14,15-LTA4 (23).
Fig. 2. UV analysis of LTA epoxide formation under biphasic
reaction conditions. The UV spectrum of 15S-HPETE substrate
(40 ␮g/ml) in 5 ml hexane was recorded before the reaction and
after vortex mixing for 90 s at 0–4°C with 1.5 mg 15-LOX-1 enzyme
in 400 ␮l 0.1 M Tris (pH 7.5).
In optimizing the conditions for the reaction of 15S-
HPETE with 15-LOX-1, we used a 12.5-fold excess of hexane
over the aqueous phase (pH held at 7.5) and a mixing
time of 90 s at 0°C. Comparison of the UV spectrum of
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Fig. 3. RP-HPLC analysis of the reaction of 15-LOX-1 with 15S-
HPETE. RP-HPLC analysis used a Waters Symmetry C18 column
(0.46 × 25 cm), a flow rate of 1 ml/min, and a solvent system of
methanol/20 mM triethylamine (pH 8.0) (90/10, by volume) with
UV detection at 270 nm.
below) helps confirm the proton assignments. The ex-
panded regions for the olefinic protons (5.0–6.5 ppm)
and the epoxide protons (2.6–3.1 ppm) illustrate the split-
ting of individual signals and associated coupling constants
from which the stereochemistry can be derived. These
provide the configuration of the double bonds, in particu-
lar identifying the conjugated triene as 8Z,10E,12E, and on
the epoxide protons the 2 Hz coupling between H14 and
H15 identifies the epoxide configuration as trans. Based
on these analyses and with the reasonable assumption that
the original 15S configuration is retained, the structure of
the epoxide product can be defined as 14S,15S-trans-ep-
oxy-eicosa-5Z,8Z,10E,12E-tetraenoate. This confirms the
structure originally proposed for this intermediate (23,
27), now named eoxin A4, precursor of the proinflamma-
tory eoxins of human eosinophils in asthma (3, 28).
Leukotriene epoxide formation from enantiomeric
HPETE substrates
To investigate the significance of substrate chirality to
LTA epoxide formation, initially we examined the reac-
tion of 15-LOX-1 with the mirror image substrate, 15R-
HPETE. The analyses showed, however, that the major
product by far is the keto derivative 15-oxo-eicosatetra-
enoic acid (data not shown). Only traces of a mixture of
very minor products exhibiting an LTA-related UV chro-
mophore were detectable.
With the requirement that the LOX enzyme used in
these preparations should have very high catalytic activity
(which our available preparations of mammalian 5-LOX
did not), we switched to the use of recombinant Arabidop-
sis AtLOX1 as the arachidonate 5-LOX (22). This enzyme
is a homolog of the potato 5-LOX (22) that is capable of
LTA epoxide synthesis and has been used as a model for
the study of 5-LOX-catalyzed leukotriene synthesis in ear-
lier studies (7, 29, 30). Although the native activity of these
plant enzymes is as a linoleic acid 9S-lipoxygenase, they
form 5S-HPETE as a prominent product from arachidonic
acid and can further convert this to LTA4 (22, 29, 30). In-
deed, after vortex mixing of AtLOX1 with 5S-HPETE at 0°C,
UV spectroscopy of the hexane phase showed a decrease in
Fig. 4. NMR analysis of 14,15-LTA4. Spectra were recorded in d6-
benzene at 283 K using a Bruker 600 MHz spectrometer. The two-
dimensional H,H-COSY spectrum is shown below; shown above is
an expanded view of the olefinic protons (5.0–6.5 ppm) and epox-
ide protons (2.6–3.1 ppm).
substrate and appearance of a new chromophore with ␭
max
at 280 nm, characteristic of LTA4 epoxide (Fig. 5A). After
methyl esterification with diazomethane, the product was
analyzed by RP-HPLC (Fig. 5B). The complete structure
was then established by 1H-NMR (see supplementary data).
Most importantly, this confirmed the 5,6-trans configura-
tion of the epoxide (J5,6 = 2 Hz) (31) (Fig. 5C).
Incubation of the 5R enantiomer of 5-HPETE with
5-LOX was performed under the same conditions. UV spec-
troscopy of the hexane phase after vortex mixing showed the
appearance of a less well-defined spectrum in the 280 nm
Structural analysis of leukotriene A epoxides
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Fig. 5. Product analyses from reactions of 5S- and 5R-HPETE with 5-LOX. A: UV spectrum of 5S-HPETE in hexane before and after vor-
tex mixing with 5-LOX enzyme (AtLOX1). B: Reversed-phase HPLC analyses of the product methyl esters from 5S-HPETE. Column: Waters
Symmetry C18, 25 × 0.46 cm; solvent, methanol/20 mM triethylamine (pH 8.0) (90/10, by volume); flow rate, 1 ml/min, UV detection at
270 nm. There was no sign on our HPLC chromatograms of a conjugated triene-containing keto derivative, a reported nonenzymatic rear-
rangement product of LTA4 epoxide (46). C: Partial 1H-NMR spectrum (2.45–3.25 ppm) of the trans-LTA epoxide product from 5S-HPETE.
D–F: The UV (D), HPLC (E), and NMR (F) analyses of 5R-HPETE reaction with 5-LOX.
region, comprised of a mixture (Fig. 5D). RP-HPLC analysis
of the esterified hexane phase demonstrated two products
of the incubation (Fig. 5E). The major product exhibited a
dienone chromophore with ␭max in MeOH/H2O (90:10) at
281 nm. Its structure was identified as 5-oxo-eicosatetra-
enoic acid (5-oxo-ETE) by GC-MS analysis of its trimethyl-
silyl ester methoxime derivative (supplementary Fig. I),
and by reduction with NaBH4 to 5-hydroxyeicosatetraenoic
acid, identified by comparison to an authentic standard.
The less abundant product displayed a conjugated triene
transformations of the natural product and leukotriene
epoxides prepared by total chemical synthesis (15–17). It
has been widely assumed that other LOX-catalyzed LTA-
type products will be the direct structural analogs of LTA4,
although the equivalent experimental support is lacking.
The possibility that LOX reactions could lead to cis-epoxy
LTA4 was deduced by Corey and coworkers (32), who
identified the stereochemistry of the initial LOX-catalyzed
hydrogen abstraction as a determinant of the trans or cis
epoxide configuration. Herein we identified the stereo-
chemistry of the HPETE substrate as an additional deter-
minant. In the following discussion we present a model
that rationalizes these observations and predicts the LTA
epoxide stereochemistry in novel LOX-catalyzed reactions.
To explain the available results, we further developed a
conceptual model underlying the mechanistic basis of the
LOX-catalyzed transformation of HPETE precursor to
LTA4-type epoxide (Fig. 7). The nonheme iron in the
LOX active site must be involved in the initial hydrogen
abstraction from the HPETE substrate and cleavage of the
hydroperoxide moiety (33, 34). Key to our thinking, there-
fore, is that the hydrogen and hydroperoxide should ex-
hibit a suprafacial relationship. Because the conversion of
[10R-3H]5-HPETE to LTA4 is associated with a primary iso-
tope effect resulting in an enrichment in the specific activ-
ity of the unreacted substrate (35–37), this provides critical
evidence identifying the 10pro-R hydrogen abstraction as
the first irreversible step in leukotriene A4 biosynthesis.
This implicates the ferric iron (hydroxide) in 5-LOX as
the active species catalyzing the first step of the transfor-
mation. The observation that reducing inhibitors of the
lipoxygenase that leave the active site iron in the ferrous state
block the dioxygenase reaction and LTA synthesis (7, 23)
chromophore with ␭
at 280 nm. The structure of this
max
LTA epoxide derived from 5R-HPETE was established by
1H-NMR (see supplementary data), most significantly de-
fining the 5,6-epoxide configuration as cis (J5,6 = 4 Hz),
thus identifying the product as the 5R,6S cis epoxide, 5-epi-
LTA4 (Fig. 5F). Its yield was similar to that of LTA4 (the
dominant recovery of 5-oxo-ETE from 5R-HPETE being in
excess of its production from 5S-HPETE). Comparison of the
NMR spectra of LTA4 and its 5-epimer also defined their
identical double bond configurations as 7E,9E,11Z,14Z
(Fig. 6A). Their UV spectra are almost superimposable,
with the difference only discernible with the two overlaid
(Fig. 6B).
DISCUSSION
The dual purposes of our study were to enable the di-
rect structural analysis of the unstable LTA-type epoxides
from lipoxygenase reactions and to further the under-
standing of factors that control their precise stereochemis-
try. The structure of the classic 5-lipoxygenase product,
LTA4, was established beyond doubt by comparison of
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Fig. 7. Lipoxygenase-catalyzed transformations to LTA epoxides.
LTA synthesis is initiated by the ferric form of the LOX enzyme
(A–C), whereas dehydration to the ketone involves the ferrous en-
zyme (D, below the dividing line).
It follows from this that the iron must have access to the
initial hydrogen abstracted and to the hydroperoxide (i.e.,
the two are on the same face of the substrate). In achieving
this, 5S-HPETE (the natural enantiomer) must assume the
transoid conformation at the 5-carbon, thus dictating
the natural trans-epoxy configuration of LTA4 (Fig. 7A).
The enantiomeric substrate, 5R-HPETE, has to assume the
cisoid conformation, which results in its transformation to
5-epi-LTA4, now established experimentally (Fig. 7B).
To complete the picture with related transformations,
Fig. 7C illustrates the result reported by Corey and cowork-
ers (32) in which an 8R-LOX activity, which catalyzes pro-S
hydrogen abstraction from C-10 (38), converted 5S-HPETE
to the cis epoxide 6-epi-LTA4 (32); by contrast, 5S-LOX,
which abstracts the 10pro-R hydrogen (30), produced trans-
epoxy LTA4 from 5S-HPETE, as expected (32). The epox-
ides were identified indirectly based on a difference in
pattern of the hydrolysis products in comparison to hydro-
lysis of the synthetic standards (32). Because it is now
known that R and S lipoxygenases are closely related en-
zymes (39, 40) and that substrate is exposed to the same
open ligand position of the active site iron (41), we de-
duce that the 5S-HPETE substrate binds in an opposite
head-to-tail orientation in 5S-LOX and 8R-LOX to present
the appropriate C-10 hydrogen for abstraction by the non-
heme iron (42). Accordingly, the substrate is shown in the
reversed orientation in the 8R-LOX reaction illustrated in
Fig. 7C. Reactions that appear feasible “on paper” may or
may not be executed favorably, depending on steric con-
siderations within the LOX enzyme. This is evident in the
reaction of 15R-HPETE with 15-LOX-1 in which LTA ep-
oxide synthesis is formed in only trace amounts. Similarly,
in the reaction of 5R-HPETE with 5-LOX, the formation
of 5-epi-LTA4 is superseded by 5-oxo-ETE synthesis as the
Fig. 6. Spectral comparison of LTA4 and 5-epi-LTA4. A: NMR spec-
tra of the olefin protons in LTA4 and 5-epi-LTA4 methyl esters. B:
Overlay of the UV spectra of LTA4 and 5-epi-LTA4 (cis-epoxy, 5R,6S).
Top: detailed view. Bottom: full spectra, 200–350 nm.
provides further support for involvement of the ferric iron
in catalyzing the initial hydrogen abstraction:
Fe3ꢀꢁ OH ꢀ HRꢁ OOH o Fe2ꢀꢁ OH2 ꢀ •ROOH
Further reaction will entail homolytic cleavage of the
hydroperoxide, thus cycling the lipoxygenase ferrous iron
back to ferric, because radical recombination produces
the epoxide product:
Fe2ꢀꢁ OH ꢀ •ROOH o Fe3ꢀꢁOH ꢀ H O ꢀ R O
ꢂ
ꢃ
2
2
Structural analysis of leukotriene A epoxides
759

Supplemental Material can be found at:
http://www.jlr.org/content/suppl/2012/12/13/jlr.M033746.DC1
.html
15. Rådmark, O., C. Malmsten, B. Samuelsson, D. A. Clark, G. Goto,
major product in catalysis initiated by the ferrous non-
heme iron (Fig. 7D) (43, 44).
A. Marfat, and E. J. Corey. 1980. Leukotriene A: stereochemistry
and enzymatic conversion to leukotriene B. Biochem. Biophys. Res.
Commun. 92: 954–961.
The concepts developed here on the determinants of cis
or trans LTA4 epoxide configuration establish a rational
mechanism underlying the structures of these key biosyn-
thetic intermediates. The concepts can be applied to the
enzymatic formation of novel LTA-type epoxides postulated
as intermediates in biosynthesis of the resolvin, protectin,
and maresin lipid mediators formed from eicosapen-
taenoic (20:5␻3) and docosahexaenoic (22:6␻3) fatty ac-
ids (4, 5). In fact, because S-lipoxygenases predominate in
higher animal biology (45) and are implicated in the syn-
thesis of the resolvin/protectin/maresin mediators, it is
possible that novel transformations involving the LTA-type
epoxides in higher animals follow the same relationships
as in the 5S-LOX pathway to the leukotrienes. Isolation
and identification of these intermediates are in progress
to examine the structures experimentally.
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The authors thank Dr. Don F. Stec for help with the NMR
analyses.
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.html
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