Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450

Article abstract of DOI:10.1194/jlr.M003061

Cytochromes P450 (CYPs) metabolize polyun-saturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., ω6 in AA or ω3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefi n of AA (ω6), EPA (ω3), or DHA (ω3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs. Copyright

Full text of DOI:10.1194/jlr.M003061

Stereoselective epoxidation of the last double bond of
polyunsaturated fatty acids by human
cytochromes P450
Danièle Lucas,1,*,†,§ Sophie Goulitquer,*,† Jan Marienhagen,** Maude Fer,† Yvonne Dreano,*,†
Ulrich Schwaneberg,** Yolande Amet,*,†,§ and Laurent Corcos*
INSERM,* U613, ECLA unit, Brest, F-29200, France; Université Européenne de Bretagne,† Faculté de
Médecine, Brest, F-29238, France; C.H.U. de Brest,§ Laboratoire de Biochimie, Brest, F-29609, France; and
RWTH Aachen University,** Worringer Weg 1, 52074 Aachen, Germany
Abstract Cytochromes P450 (CYPs) metabolize polyun-
saturated long-chain fatty acids (PUFA-LC) to several classes
of oxygenated metabolites. Through use of human recombi-
nant CYPs, we recently showed that CYP1A1, -2C19, -2D6,
-2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2,
-2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic
acid (AA), eicosapentaenoic acid (EPA), and docosa-
hexaenoic acid (DHA), respectively. It is worth noting that
the last double bond of these PUFAs, i.e., ␻6 in AA or ␻3 in
EPA and DHA, respectively, was preferentially epoxidized.
In this study, we have characterized the stereoselectivity of
this epoxidation reaction by comparison with the PUFA-LC
epoxide stereoisomers obtained from the enantioselective
bacterial CYP102A1 F87V. The stereoselectivity of the ep-
oxidation of the last olefin of AA (␻6), EPA (␻3), or DHA
(␻3) differed between the CYP isoforms but was similar for
EPA and DHA. These data give additional insight into the
PUFA-LC epoxide enantiomers generated by the hepatic
CYPs.—Lucas, D., S. Goulitquer, J. Marienhagen, M. Fer, Y.
Dreano, U. Schwaneberg, Y. Amet, and L. Corcos. Stereose-
lective epoxidation of the last double bond of polyunsatu-
rated fatty acids by human cytochromes P450. J. Lipid Res.
2010. 51: 1125–1133.
family 1 to 3 are mainly epoxygenases, whereas CYP iso-
forms from family 4 are mainly ␻-hydroxylases (4, 5).
CYP epoxygenases convert arachidonic acid (AA) to four
epoxyeicosatrienoic acid regioisomers (5,6-, 8,9-, 11,12-,
and 14,15-EET) that function as lipid mediators. EETs pro-
duce vascular relaxation, have antiinflammatory effects
on blood vessels and in the kidney, promote angiogenesis,
and protect ischemic myocardium and brain [for review,
see (6–8)]. CYP epoxygenases also convert the ␻3-PUFA
eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) to epoxy-derivatives (9), which are potent dilators of
coronary arterioles (10–12) or pulmonary artery (13) and
inhibit platelet aggregation (14). The EPA-derived epox-
ides account for five epoxyeicosatetraenoic acids (5,6-, 8,9-,
11,12-, 14,15-, and 17,18-EETeTr), whereas the DHA-derived
epoxides account for six epoxydocosapentaenoic acids
(4,5-, 7,8-, 10,11-, 13,14-, 16,17-, and 19,20-EDP). A high
regio- and stereoselectivity has been observed when testing
the effects of chemically synthesized epoxides from AA and
EPA. For example, in the rat, renal arteries were dilated by
11(R),12(S)-EET but not by 11(S),12(R)-EET or 14,15-EET
enantiomers (15). Also in rats, among all EETeTr enantio-
mers, only the 17(R),18(S)-enantiomer, not 17(S),18(R),
was effective on calcium-activated potassium (BK) channels
in isolated cerebral arteries (11). However, in porcine coro-
nary microvessels, all regioisomeric EETeTrs had similar
Supplementary key words regioselectivity • arachidonic acid • ei-
cospentaenoic acid • docosahexaenoic acid • CYP102A1 F87V
Cytochromes P450 (CYPs) belong to a protein super-
family among which some members metabolize polyun-
saturated long-chain fatty acids (PUFA-LC) to several
classes of oxygenated metabolites. The product profiles
depend on the involved CYP isoforms and may consist of a
series of regio- and stereo-isomeric epoxides and hydrox-
ylated compounds (1–3). In humans, CYP isoforms from
Abbreviations: AA, arachidonic acid [5,8,11,14-eicosatetraenoic
acid or C20:4, (n-6)]; BK, calcium-activated potassium CYP, cytochrome
P450; EET, epoxieicosatrienoic acid; EPA, eicosapentaenoic acid
[5,8,11,14,17-eicosapentaenoic acid or C20:5, (n-3)]; EETeTr, epoxi-
eicosatetraenoic acid; DHA, docosahexaenoic acid [4,7, 10,13,16,19-
docosahexaenoic acid or C22:6, (n-3)]; EDP, epoxidocosapentaenoic
acid; F87, phenylalanine 87; PUFA-LC, polyunsatured long-chain fatty
acids; DBA, n-dibutylamine; OR, cytochrome P450 reductase; m-CPBA,
m-chloroperbenzoic acid; LC-MS-APCI, liquid chromatography-mass
spectrometry-atmospheric pressure chemical ionization; V, valine.
1 To whom correspondence should be addressed.
This work was supported by a fellowship from the Ministère de l’Enseignement
Supérieur et de la Recherche (to S.G.).
Manuscript received 21 November 2009 and in revised form 24 November 2009.
Published, JLR Papers in Press, November 24, 2009
DOI 10.1194/jlr.M003061
e-mail: daniele.lucas@univ-brest.fr
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 51, 2010
1125

from BD-Biosciences Gentest (Supersome Enzymes, Woburn,
MA). BD CYP Supersomes are recombinant cDNA-expressed CYP
enzymes prepared from the baculovirus-infected insect cell sys-
tem BTI-TH-5B1-4. Their respective characteristics were as follows
(data from the suppliers): CYP1A1: 105 pmol/mg protein, NADPH
P450 reductase 2400 nmol/min.mg, and 7-ethoxyresorufin deeth-
ylase activity 39 minϪ1; CYP1A2: 159 pmol/mg protein, NADPH
P450 reductase 2600 nmol/min.mg, and phenacetin deethylase
activity 56 minϪ1; CYP2C8: 278 pmol/mg protein, NADPH P450
reductase 170 nmol/min.mg, b5 410 pmol/mg, and paclitaxel
6-␣-hydroxylase activity 9.9 minϪ1; CYP2C9: 370 pmol/mg protein,
NADPH P450 reductase 260 nmol/min.mg, b5 560 pmol/mg, and
diclofenac 4’-hydroxylase activity 20 minϪ1; CYP2C19: 192 pmol/
mg protein, NADPH P450 reductase 880 nmol/min.mg, b5 260
vasodilatory potencies (16). Thus, the regio- and stereo-
chemical features of the CYP-derived metabolites govern
their biological activity and/or potency and functional
significance. The biological activity of DHA epoxides regio-
isomers and enantiomers is still largely unknown as are the
identity and reaction specificity of the individual CYP iso-
forms involved in EPA and DHA metabolisms (17).
According to previous studies, CYP isoforms display dif-
ferent regioselectivities for PUFA hydroxylation and ep-
oxidation. For example, CYP1A1 is mainly an hydroxylase
toward AA, whereas it acts primarily as an epoxygenase to-
ward EPA as substrate (18). CYP102A1 (Bacillus Megaterium
BM-3), a bacterial CYP that closely resembles eukaryotic
microsomal P450s (19), is mainly an ␻-hydroxylase for
AA and a highly specific epoxygenase for EPA. Replace-
ment of phenylalanine 87 (F87) with valine (V) converts
CYP102A1 into a highly specific AA epoxygenase (20).
Through use of human recombinant CYPs, we recently
evidenced that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are
mainly hydroxylases of AA, EPA, and DHA, whereas
CYP2C8, -2C9, -2J2, and -1A2 are mainly epoxygenases (21).
Epoxygenase regioselectivity depends on the nature of the
unsaturated fatty acid of concern, because four, five, or six
double bonds can be epoxidized. The number of epoxyge-
nase products increases with the number of unsaturations,
and the last double bond is preferentially oxidized, i.e., ␻6
in AA or ␻3 in EPA and DHA, respectively. However, to our
knowledge, the studies about the stereoselectivity of this
epoxidation have only dealt with AA or EPA and a few CYPs
(18, 20, 22–29); none of them has been focused on DHA.
Therefore, we have systematically investigated the stereo-
chemistry of the epoxidation reaction of the last double
bond of AA, EPA, and DHA by nine recombinant CYPs,
with special emphasis on DHA epoxide enantiomers. The
CYP102A1 F87V mutant was thus used as a model because
of its high enantiofacial selectivity; indeed, it is known to
epoxidize the si,re face of the last olefin of AA or EPA. This
allowed us to identify, by comparison, the PUFA epoxide
enantiomers generated by the human recombinant CYPs.
pmol/mg, and S-mephenytoin 4’-hydroxylase activity 21 minϪ1
CYP2D6: 126 pmol/mg protein, NADPH P450 reductase 3500
nmol/min.mg, and bufuralol 1’-hydroxylase activity 59 minϪ1
;
;
CYP2E1: 182 pmol/mg protein, NADPH P450 reductase 4100
nmol/min.mg, b5 700 pmol/mg, and p-nitrophenol hydroxylase
activity 11 minϪ1; CYP2J2: 278 pmol/mg protein, NADPH P450
reductase 450 nmol/min.mg, b5 320 pmol/mg, and terfenadine
hydroxylase activity 43 minϪ1; CYP3A4: 870 pmol/mg protein,
NADPH P450 reductase 2800 nmol/min.mg, b5 1200 pmol/mg,
and testosterone 6-␤-hydroxylase activity 150 minϪ1
.
CYP102A1 F87V (P450 BM-3 F87V)
Crude extracts of CYP102A1 F87V were prepared according to
(30). In brief, 250 ml of TBamp media, supplemented with 250 µl
trace element solution, was inoculated with an LBamp-overnight
culture of Escherichia coli DH5a pCWOri P450 BM-3 F87V. After
reaching an optical density at 578 nm of 0.8 during cell cultiva-
tion (30°C, 250 rpm), D-aminolevulinic acid as precursor of por-
phyrin synthesis (0.5 mM, final concentration) was added and
expression was induced by supplementation with isopropyl-beta-
D-thiogalactoside (ITPG) (100 mM in final concentration). After
20 h of expression, E. coli cells were harvested by centrifugation
and resuspended in phosphate buffer (25 ml; KH2PO4/K2HPO4,
25 mM, pH 7.5). The E. coli cells were subsequently lysed using a
high-pressure homogenizer (1500 bar, three cycles). The lysate
was centrifuged and further clarified by filtration through a low
protein binding filter (0.45 mm). The resulting crude extract was
lyophilized overnight and the powder stored at – 20°C. CYP102A1
F87V concentrations were estimated to be about 20 µM from CO-
difference spectra as reported by Omura and Sato(31) using ␧ =
91 mMϪ1. cmϪ1. Use of the NADPH-depletion assay according to
a well-established protocol led to 2.57 U.mgϪ1 as activity of this
mutant in the crude extract when lauric acid was used as sub-
strate (32).
EXPERIMENTAL PROCEDURES
Chemicals
All chemicals of analytical grade were from Sigma-Aldrich
(L’Isle d’Abeau, France). HPLC-purity grade solvents were from
Carlo Erba (Val de Reuil, France). The counter-ion for HPLC,
n-dibutylamine in its acetate concentrated form and m-chloro-
perbenzoic acid (m-CPBA), were both from Fluka (Buchs, Swit-
zerland). The following PUFAs were from Cayman Chemicals
(Spi-Bio, Montigny le Bretonneux, France): 5Z,8Z,11Z,14Z--
eicosatetraenoic acid (AA C20:4), 5Z,8Z,11Z,14Z,17Z-eicosapen-
taenoic acid (EPA C20:5), 4Z,7Z,10Z,13Z,16Z,19Z-DHA C22:6.
[1-14C]AA (2.07 GBq/mol) was from Amersham Biosciences
(UK), [1-14C]EPA (1.92 GBq/mol) was from MP Biomedicals
(Irvine, CA), and [1-14C]DHA (1.92 GBq/mol) was from Moravek
Biomedicals (Brea, CA).
Chemical synthesis of radiolabeled epoxides
14C-radiolabeled epoxides of PUFA were synthetized with
m-CPBA then separated by HPLC and purified. Briefly, 100
nmol of [1-14C]PUFA were incubated with one equivalent of
m-CPBA diluted in 0.1 ml of CH2Cl2 for 15 min at room tempera-
ture. Samples were then dried under nitrogen, resuspended in
ethanol, and separated by HPLC as described below. The radio-
labeled monoepoxides, previously identified by LC-MS (9), were
collected.
Synthesis of radiolabeled epoxides by CYPs
All of the reaction mixtures were prepared in a final volume of
100 µL and contained 1 nmol of radiolabeled substrates, [1-14C]
AA, [1-14C]EPA, and [1-14C]DHA, in 0.12 M phosphate buffer,
pH 7.4 containing 5 mM MgCl2 and 10 pmol of recombinant
CYP. To obtain the final concentration of 10 µM (CYP2C9, 2C8,
Recombinant CYPs and CYP102A1 F87V
Microsomes containing the human recombinant CYPs
(CYP1A1, -1A2, -2C8, -2C9, -2C19, -2E1, -2D6, -2J2, and -3A4) were
1126
Journal of Lipid Research Volume 51, 2010

CYP2C19, CYP2J2) or 50 µM (CYP1A1, 1A2, 2E1, 3A4), these
compounds were supplemented with cold substrate. After a 5
min preincubation at 37°C, the reaction was started by addition
of 1 mM NADPH. Different substrate concentrations were used
to avoid auto-inhibition previously described (21). After 30 min,
the reaction was stopped by addition of 0.1 ml acetonitrile con-
taining 0.2% acetic acid (v/v). Fatty acids and their metabolites
were twice extracted by 2 ml ethyl acetate. Samples dried under
nitrogen were dissolved in 50 µL ethanol, and the metabolites
were resolved by HPLC as described below.
flow rate of 1 ml/min. The enantiomers were detected with
a ␤-radiometric flow scintillation analyzer Flo-one 500Tr
(Perkin-Elmer-Packard Biosciences). The enantiomers were
identified by comparison with known products generated by
CYP102A1 F87V catalyst.
GC-MS identification of epoxides
The epoxidized samples were analyzed by GC-MS on an Agi-
lent GC 6890 coupled to a 5973 MS Detector (Agilent, Les Ulis,
France) and equipped with a DB-5MS column (low-bleeding,
5% phenyl/95% dimethylpolysiloxane phase) 30 m × 0.25 mm
inner diameter × 0.25 µm film thickness (J and W Scientific, Agi-
lent). The temperatures of the injection port and interface were
250 and 280°C, respectively; those of the ion source and MS
analyzer were set at 150 and 106°C, respectively. The samples
were injected in splitless mode. The oven temperature was
first set at 60°C for 5 min, elevated to 230°C at the rate of
50°C/min and then to 290°C at 1°C/min. The compounds
were ionized by electron impact at 70 eV energy. The carbox-
ylic group of DHA monoepoxides was converted to methyl
ester as previously described. The reaction solvent was dried
under a nitrogen stream and redissolved in hexane prior to
injection. Analytes were detected by total ion current from
m/z 50 to 600.
Incubation with CYP 102A1 F87V
The reaction mixture contained 100 µM of AA, EPA, or DHA
and radiolabeled substrate, 10 µg of crude extract, 0.12 M phos-
phate buffer, pH 7.4, and 5 mM MgCl2. After a 2 min preincuba-
tion at 30°C, the reaction was started by addition of 1 mM
NADPH. After 2 min, the reaction was stopped by addition of 0.1
ml of a solution of acetonitrile with 0.2% acetic acid (v/v). The
samples were then processed as above.
HPLC conditions
The monoepoxide or hydroxylated metabolites of fatty acids
were separated by a reversed phase ion-pairing LC as previously
reported (9). Briefly, the regioisomers were separated isocrati-
cally on a RP-Capcell Pak C18, 120 Å, 250 mm × 4.6 mm, particle
diameter 5 µm (Shiseido Co Ltd, Japan, distributed by C.I.L, Ste
Foy La Grande, France). The mobile phase A consisted of a mix-
ture of water-methanol-acetonitrile (52/8/40; v/v/v) containing
15 mM n-dibutylamine. The composition of this mobile phase
was adjusted for each substrate as previously reported (9). Radio-
activity was measured with a ␤-radiometric flow scintillation ana-
lyzer Flo-one 500Tr (Perkin-Elmer-Packard Biosciences) with
Ultima Flo scintillation cocktail added to the mobile phase at a
flow of 1 ml/min. The radiolabeled monoepoxides, previously
identified by LC-MS, were collected. The samples acidified by 20
µL of acetic acid were twice extracted by 2 ml ethyl acetate and
dried under nitrogen. Then, the methylation reaction was started
by addition to the samples of 300 µL of diethylether saturated
with diazomethane. After incubation for 15 min at room tem-
perature, the samples were dried under nitrogen and redissolved
into hexane prior to analysis by chiral HPLC.
RESULTS
Regioselectivity of CYP isoforms and CYP 102A1 F87V
on the epoxidation of AA, EPA, and DHA
Incubation of AA, EPA, or DHA with recombinant
CYP1A1, -1A2, -2C8, -2C9, -2C19, -2D6, -2E1, -2J2, and
-3A4 resulted in the preferential epoxidation of the last
double bond of the alkyl chain and generated the 14,15-
EET, 17,18- EETeTr, and 19,20-EDP, respectively (Table 1).
A high regioselectivity was observed with CYP1A1, -2D6,
and -2E1 as epoxidation took place mainly on the last and
the penultimate double bonds. Such a regioselectivity
had been already reported with CYP102A1 or its mutants
toward AA and EPA (20, 24). However, to our knowl-
edge, no description of DHA metabolism by CYP102A1 or
its F87V mutant is available in the literature. Thus, this
␻3-PUFA was incubated with the CYP102A1 F87V crude
extract to generate the corresponding epoxides (EDPs).
After separation by reversed-phase ion-pair HPLC, the
main metabolites appeared to be two monoepoxides in
the ratio 37:63 and identified as the 16,17- and the 19,20-
EDP (Fig. 1A). Their characterization relied on their
HPLC retention times identical to those of synthetic
16,17- and 19,20-EDP (Fig. 1B) and on their mass spectra
analysis by LC-MS-APCI (Fig. 1C) or GC-MS-electron ion-
ization after derivatization as methyl esters. LC-MS-APCI
analysis showed the presence of major negative fragment
ions at m/z 343 [base peak, M-H]Ϫ. The fragmenta-
tion of regioisomers was governed by the epoxide ring
located in the alkyl chain and involved either a cleavage
in position ␣ with respect to the epoxide moiety or a
cleavage of the oxirane ring itself at two positions. This
led to three characteristic ions from 16,17-EDP, namely
m/z=233 and 273 (fragmentation in the ␣ position) and
m/z=245 (fragmentation in the oxirane ring). Two char-
HPLC-MS identification of epoxides
The epoxidized metabolites were separated under the HPLC
conditions described above and identified by MS on a mass
spectrometer (Navigator model, Thermo-Electron, Les Ulis,
France) equipped with an atmospheric pressure chemical ion-
ization (APCI) source and operated in negative-ion mode. The
cone voltage was optimized in the range of 30–45 V to enhance
molecular fragmentation. The vaporizer temperature for APCI
was 350°C and the source was 150°C. The drying nitrogen flow
was 352 L/h, the needle of the corona pin was set at 3 kV, the
skimmer at 0.9 V, and the quadrupole ion energy at 2.6 V. For
epoxide identification, the negative ions were monitored in full
scan mode in 1 s from m/z 50 to 600.
HPLC chiral analysis
For chiral analysis, the standard reactions of incubation
were repeated 3- to 7-fold. The products collected from the
HPLC runs, i.e., 14,15-EETs, 17,18-EETeTr, or 19,20-EDP,
were pooled and derivatized with diazomethane to methyl-
esters that were subsequently analyzed by chiral-phase HPLC
on a Chiralcel OB column (250 × 4.6 mm; Daicel, Interchim,
Montluçon, F). The solvent was hexane with 0.3% isopropyl
alcohol and 0.05% (v/v) acetic acid and was delivered at a
Stereoselective epoxidation of polyunsatured fatty acids
1127

TABLE 1. Percentage of each epoxide formed from three PUFAs by CYPs
Epoxides
Last double bond:
14,15- EET
17,18-EETeTr
19,20-EDP
Penultimate bond:
11,12-EET
14,15-EETeTr
16,17-EDP
Ante-Penultimate bond:
8,9-EET
11,12-EETeTr
13,14-EDP
5,6-EET
8,9-EETeTr
10,11-EDP
5,6-EETeTr
7,8-EDP
CYP
1A1
PUFA
4,5-EDP
AA
100
100
100
26
76
54
54
41
47
56
35
15
37
56
27
47
100
100
100
74
nd
nd
nd
43
9
nd
nd
nd
10
9
nd
nd
nd
21
6
—
nd
nd
—
nd
6
—
nd
nd
—
8
—
—
nd
—
—
nd
—
—
nd
—
—
nd
—
—
nd
—
—
nd
—
—
nd
—
—
nd
—
—
nd
—
—
nd
EPA
DHA
AA
EPA
DHA
AA
EPA
DHA
AA
EPA
DHA
AA
EPA
DHA
AA
EPA
DHA
AA
EPA
DHA
AA
EPA
DHA
AA
1A2
2C8
2C9
2C19
2D6
2E1
2J2
22
46
40
18
31
25
25
12
17
15
18
nd
nd
nd
15
7
8
10
nd
nd
20
nd
15
42
11
14
32
10
nd
nd
nd
7
nd
nd
15
8
13
7
nd
19
15
13
17
12
40
8
6
—
nd
14
—
nd
nd
—
nd
nd
—
3
12
25
nd
nd
nd
10
nd
nd
15
8
21
20
18
nd
nd
nd
93
40 a
16
nd
37
18
18
<1
27 b
37
60 a
50
7
—
4
77
29
41
3A4
EPA
DHA
AA
EPA
DHA
41
16
nd
nd
nd
7
99 b
73 c
63
CYP102A1
(F87V)
—
nd
nd
The major epoxide is highlighted. nd: not detectable. Values represent mean of two separate experiments.
a (21).
b (24).
c (20).
acteristic ions were obtained from 19,20-EDP, namely
m/z=71 (fragmentation in the ␣ position) and m/z=285
(fragmentation in the oxirane ring). Other ions, i.e., m/z
325[M-H2O]Ϫ, m/z 299 [M-CO2]Ϫ, and m/z 281 [M-H2O-
CO2]Ϫ, were issued from fragmentation of the carbox-
ylic group and were common for 16,17- or 19,20-EDP
regioisomers. Mass spectra analysis by GC-MS-electron
ionization after derivatization of the two monoepoxides
as methyl esters showed major fragment ions at m/z 358
(base peak), m/z 289 and 247 (fragmentation in the ␣
position), and m/z 260 (fragmentation of oxirane ring)
for 16,17-EDP. Characteristic ions for 19,20-EDP were at
m/z 287 and 329 (fragmentation in the ␣ position) and
m/z 300 (fragmentation of oxirane ring). These mass
spectra were identical to those generated under similar
conditions by synthetic 16,17- or 19,20-EDP regioisomers
(data not shown).
However, the stereoselectivity of the epoxidation
reaction by CYP isoforms is poorly known, especially
when DHA is a substrate. The lack of commercially
available enantiomeric standards for EDP prevents their
easy characterization. This led us here to identify these
enantiomers from the well-known stereoselectivity of
CYP102A1 F87V (24).
Stereoselective epoxidation of the last double bond of
AA and EPA by CYP102A1 F87V and recombinant human
CYPs
According to previous results (24), incubation of
CYP102A1 with AA generates 18(R)-OH-AA and
14(S),15(R)-EET in a 4:1 molar ratio, whereas EPA is me-
tabolized to 17(S)18(R)-EETeTr with 97% optical purity
as the sole reaction product. Conversely, the metabolism
of PUFAs by the CYP102A1 F87V mutant generates
14(S),15(R)-EET (99% of total products) from AA and
14(S),15(R)- and 17(S),18(R)-EETeTr, 26 and 69% of to-
tal, respectively, from EPA (20). In this study, the crude
extract of CYP102A1 F87V yielded the same main regioiso-
mers as those obtained with purified CYP102A1 F87V pro-
tein, i.e., 14,15-EET for metabolism of AA and a mixture of
14,15- and 17,18-EETeTr for EPA (not shown). These re-
gioisomers were known to be the S,R enantiomers (20).
Their use as standards enabled us to identify unambigu-
ously the optical isomers formed by the epoxidation of the
last double bond of AA (␻6) and EPA (␻3) by the human
recombinant CYPs.
As shown in Fig. 2A, CYP1A1, -1A2, -2E1, -2C9, -2J2,
and -3A4 displayed a nearly racemic epoxidation of the
last double bond of AA (␻6) compared with an absolute
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Journal of Lipid Research Volume 51, 2010

Fig. 1. DHA metabolism by CYP102A1 F87V. 1A: RP-HPLC Chromatographic resolution of the products generated by CYP102A1 F87V
during the metabolism of DHA; Peak 1: 19,20-EDP; Peak 2: 16,17-EDP. A total of 100 µM of [1-14C] DHA was incubated with10 µg of crude
extract CYP102A1 F87V. The HPLC analysis was carried out as described in Experimental Procedures. 1B: RP-HPLC chromatographic reso-
lution of the products generated by chemical epoxidation of DHA; Peak 1: 19,20-EDP; Peak 2: 16,17-EDP; Peak 3: 13,14-EDP; Peak 4: 10,11-
EDP; Peak 5: 7,8-EDP. These peaks were previously identified by LC-MS (9). 1C: LC-MS of 16,17- and 19,20-EDP generated by incubation
of DHA with CYP102A1 F87V as described in Experimental Procedures. Only significant fragments of the epoxide position in the alkyl
chain were indicated.
stereoselectivity for CYP2C19, -2D6, and CYP102A1
F87V, which was in favor of the 14(R),15(S)-EET for
CYP2C19 and of its enantiomer for CYP2D6 and
CYP102A1 F87V. By contrast, epoxidation of the last
double bond of EPA (␻3) (Fig. 2B) by most of the re-
combinant CYPs generated an enantiomeric excess of
17(R),18(S)-EETeTr,whileCYP2D6,-2C8,andCYP102A1
F87V displayed an opposite stereoselectivity compared
with the other CYPs. The epoxidation stereoselectivity of
the EPA ␻3 olefin by CYP1A2, -2E1, and CYP102A1 F87V
(although opposite) was nearly absolute. As already de-
scribed (22), CYP2C8 showed an opposite stereoselectiv-
ity of epoxidation of the AA ␻6- and EPA ␻3-olefins, as
the R,S enantiomer was mainly generated from AA and
S,R from EPA.
Stereoselective epoxidation of the last double bond of
DHA by CYP102A1 F87V and human recombinant CYPs
As expected, the biosynthesis of 19,20-EDP by CYP102A1
F87V was highly asymmetric and presumably generated
the 19(S),20(R)-EDP enantiomer (87% optical purity)
(Fig. 3).
Stereoselective epoxidation of polyunsatured fatty acids
1129

Fig. 2. Stereochemistry of epoxidation of the last double bond of
AA and EPA by CYPs. Results are expressed as percentages of the
two enantiomers generated by incubation of AA (A) and EPA (B)
with CYP102A1 F87V and human recombinant CYPs. Enantiomers
were identified by comparison with the S,R enantiomer generated
by CYP102A1 F87V. * CYP2J2 data were previously reported (42).
Identification of this enantiomer was based on the
previously described C-H acceptor chemistry and active
site-binding coordinates of CYP102A1 F87V known to
preferentially deliver oxygen to the si,re-face of the last
olefinic bond for both AA (␻6) and EPA (␻3) (24). If the
enantiomer generated from DHA by CYP102A1 F87V is the
19(S),20(R)-EDP, the main product issued from epoxida-
tion of the last double bond ␻3 of DHA by CYP1A1, -1A2,
-2E1, -2C19, -2J2, and -3A4 should be the 19(R),20(S)-EDP.
Figure 4 shows that all the CYPs under study generated
an enantiomeric excess in favor of the 19(R),20(S)-EDP
for CYP1A1, -1A2, -2E1, -2J2, and -3A4 and of its optical
antipode for CYP2D6, -2C8, and CYP102A1 F87V. One
should note that the stereoselectivity displayed by CYP1A2,
-2E1, and CYP102A1 F87V was nearly absolute and the
major EDP enantiomer generated by CYP2C8 was the S,R
enantiomer. The same observations being valid for EPA
suggests that the stereoselectivity of epoxidation of the last
olefinic bond ␻3 of EPA and DHA by CYPs is very similar.
Fig. 3. HPLC chiral analysis of 19,20 EDP. Representative HPLC
chiral analysis illustrating the stereoselectivity of epoxidation of
CYP102A1 F87V and CYP1A2 during the 19,20-double bond epoxi-
dation of DHA versus the racemisation of this olefin by chemical
epoxidation.
and DHA metabolisms (17). The use of human recom-
binant CYPs allowed us to recently demonstrate that the
epoxygenase-hydroxylase activity ratio is dependent
upon the nature of PUFAs and CYP isoforms (21). How-
ever, with EPA and DHA, epoxygenase products were
more abundant than with AA. Furthermore, the last
double bond of the alkyl chain of AA (␻6), EPA (␻3), or
DHA (␻3) was preferentially epoxidized. After a reas-
sessment of the regiospecificity of AA-, EPA-, and DHA-
epoxidation of the last olefinic bond by human CYPs of
families 1–3, we have characterized the stereochemistry
DISCUSSION
Little is known about the identity and reaction speci-
ficity of the individual CYP isoforms involved in EPA
1130
Journal of Lipid Research Volume 51, 2010

by CYP102A1 F87V generated a mixture of two regioiso-
mers, namely the 14,15- and 17,18- EETeTr for EPA, as
previously described (20), or the 16,17- and 19,20- EDPs
for DHA. Thus, it appeared that the regioselectivity of the
last olefin epoxidation was very similar for EPA and DHA,
as the ␻3 and ␻6 olefins were epoxidized in each case.
Furthermore, it must be underlined that the preferred site
of AA and EPA for catalyzed oxygen insertion by CYP102A1
is the (␻-2) carbon atom leading to 18-OH-AA and
17,18-EETeTr, respectively. This indicates that both fatty
acids likely occupy more or less similar spatial coordinates
in the enzyme active site.
Modeling experiments provide evidence for lowest inter-
action energies of the C20 fatty acid-CYP1A1 complexes
when the substrates are docked in orientations leading to
hydroxylation at C19 or electron abstraction at C18 and
C17 (18). Such energetic differences may explain the
regioselectivity of CYP-mediated reactions (36). It appears
that flexible AA and EPA are stabilized in the active site
mainly by hydrophobic interactions, thus governing the
regiospecificity of substrate oxidation.
Fig. 4. Stereochemistry of epoxidation of the last double bond of
DHA by CYPs. Results are expressed as percentages of the two
enantiomers generated by incubation of DHA with CYP102A1 F87V
and human recombinant CYPs. Enantiomers were identified by
comparison with the S,R enantiomer generated by CYP102A1
F87V.
Stereoselectivity of epoxidation
of this reaction with special emphasis on DHA-epoxide
enantiomers.
The stereoselectivity of PUFA epoxidation was studied
using CYP102A1 F87V as a model because of its high
enantioselectivity. Regardless of the fatty acid, the si,re
faces of the last double bond, 14,15 or 17,18-olefin for AA
and EPA, respectively, were always selected for epoxida-
tion (20). We assumed that epoxidation would also take
place on the si,re face of the 19,20-olefin of DHA, given the
similar regioselectivity established between EPA and
DHA.
The absolute configurations of the regioisomers
were assigned by chromatographic comparisons with
enantiomerically pure methylated fatty acid epoxides
prepared by total chemical synthesis (racemic mixture)
and enzymatic epoxidation from the enantioselective
CYP102A1 F87V. Furthermore, the chiral analysis by HPLC
on Chiralcel OB showed that the S,R enantiomer epoxide
of the last double bond of AA and EPA was eluted after its
optical antipode, as was the case in all previous studies (18,
22, 23, 33, 37). These results were verified with DHA epox-
ide enantiomers, namely the 19,20-EDP. It is worth under-
lining that the elution order was the opposite on Chiralcel
OD (38) or Chiralcel OJ (28).
Our investigations showed that the epoxidation of
the last double bond by most of the hepatic CYPs is
more stereoselective with the (␻3) PUFA (EPA or DHA)
as substrates than with the (␻6) AA. Interestingly,
CYP2C8 showed an opposite stereoselectivity of this re-
action between AA and EPA or DHA. CYP2C9 epoxi-
dized EPA with a lower regioselectivity for the last olefin
than CYP2C8 as already reported (22).The same was ob-
served with DHA, resulting in a decreased capacity of
CYP2C9 to synthesize the 19,20-EDP. This prevented us
from characterizing the enantioselectivity of this
reaction.
Regioselectivity of epoxidation
Depending on the nature of the PUFAs, four, five, or six
double bonds may be concerned by the regioselectivity of
the epoxidation reaction. In this study, the preferentially
oxidized double bond by CYP1A1, -1A2, -2D6, -2J2, and
-2E1 was always the last one, i.e., 14,15 (AA), 17,18 (EPA),
or 19,20 (DHA). This high regioselectivity involves a strict
position of PUFAs in their active sites with the last double
bond close to the ferryl-oxo complex of CYPs. However,
many other double bonds have been shown to be epoxi-
dized, particularly by CYP2C enzymes (33). Recently
published crystal structures of CYP2C8/2C9 (34) have evi-
denced a great flexibility in the conformation of the active
site cavity, which allows the binding of ligands in multiple
positions. Furthermore, thanks to their high flexibility,
fatty acids would move in the close vicinity of the ferryl-oxo
complex to be epoxidized in many positions.
A comparison of the catalytic specificities of CYP102A1
and human CYP1A1 (24) also allowed some interesting
parallels. Much of what we know regarding CYP structure-
function relationships derives from studies with soluble
bacterial CYPs, especially CYP102A1 (P450 BM-3). The
crystal structure of the substrate-free heme domain re-
vealed an open access channel from the protein surface
deep into the heme active site (35). The phenylalanine
F87 is located over the heme surface in a position to an-
chor the hydrocarbon tail of substrates. Replacement of
the aromatic ring of F87 by a valine in the F87V mutant
allowed further displacement of the fatty acid molecule
along the longitudinal axis of the access channel (20).
Thus, the metabolism of AA by CYP102A1 leads to the pro-
duction of 18-OH-AA (main product) and 14,15-EET,
whereas the F87V mutant generates 14,15-EET as the sole
reaction product. In our study, metabolism of EPA or DHA
This study confirmed the previously described stereose-
lectivity of epoxidation catalyzed by the CYP2C (33, 39)
Stereoselective epoxidation of polyunsatured fatty acids
1131

and CYP1A1 (18) subfamilies in AA and EPA metabolisms.
Whereas CYP102A1 F87V is known to epoxidize the si,re
face of the last double bonds of AA, EPA, and DHA,
CYP1A1, -1A2, -2E1, -2C19, and -2J2 proved to preferen-
tially oxidize the re,si face during the metabolism of EPA or
DHA. This difference suggests that these ␻3-PUFAs were
strictly positioned in the active site with the re,si face set in
the close vicinity of the ferryl-oxo complex of these CYPs.
In our opinion, this positioning is governed by hydropho-
bic interactions but not by the reactivity of the double
bond hydrogens. In the case of CYP102A1, it has been sug-
gested that the F87 residue blocks the ␻-terminus of fatty
acids (18) so that the si,re face is presented near the ferryl-
oxo complex. By analogy, the F equivalent residues of
CYP1A1, -1A2, -2E1, and -2C19 might play the same role in
blocking the ␻-methyl terminus but in such a position that
the re,si face of the double bond will be preferentially
epoxidized.
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CONCLUSION
A careful examination of the epoxidation reaction of
the last double bonds of the three substrates emphasizes
high similarities in the stereoselectivity of EPA and DHA
epoxidation by the nine CYPs but noteworthy differences
between the CYPs. The similarities are likely attributable
to the position of the last double bond in such long-chain
fatty acids; indeed, EPA and DHA belong to the ␻3 class,
whereas AA is a member of the ␻6 class. As attempts to get
crystallized PUFA-bound human CYPs have been unsuc-
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because the enantioselective metabolism of PUFAs ap-
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conformation.
The CYP-dependent metabolism of EPA and DHA is a
source of physiologically active compounds that may con-
tribute to the beneficial cardiovascular effects attributed
to diets rich in ␻3-PUFAs (40). The relative importance
of the hepatic CYPs to biologically active metabolites in
the vasculature and other tissues remains to be estab-
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known to be involved in the regulation of blood pressure
(41), some functional effects of supplementation with ␻3-
PUFAs may result from the conversions of EPA and DHA
to the corresponding epoxidized metabolites and per-
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Stereoselective epoxidation of polyunsatured fatty acids
1133