structural and mechanistical studies on hydroperoxide conversions
TRANSCRIPT
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Structural and mechanistic studies of hydroperoxide conversions catalyzed by a CYP74 clan epoxy alcohol synthase from amphioxus (Branchiostoma floridae)
Mats Hamberg1, Bulat I. Khairutdinov3, Julia Scholz2, Florian Brodhun2, Ellen Hornung2, Ivo
Feussner2, and Alexander N. Grechkin3
1 Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, SE-17177 Stockholm, Sweden; 2 Georg-August-University, Albrecht von-Haller
Institute for Plant Sciences, Department of Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37075
Göttingen, Germany; 3 Kazan Institute of Biochemistry and Biophysics, Russian Academy of
Sciences, P.O. Box 30, 420111 Kazan, Russia
Running title: Studies on amphioxus epoxy alcohol synthase
Corresponding author:
Mats Hamberg
Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, SE-17177 Stockholm, Sweden
Tel. 46 8 52587640; E-mail [email protected]
Footnote: Abbreviations: AOS, allene oxide synthase; DES, divinyl ether synthase; EAS, epoxy
alcohol synthase; HAS, hemiacetal synthase; GC-MS, gas-liquid chromatography-mass spectrometry;
HODE, hydroxyoctadecadienoic acid; HPODE, hydroperoxyoctadecadienoic acid; HPOTrE,
hydroperoxyoctadecatrienoic acid; MC, (-)-menthoxycarbonyl; TMS, trimethylsilyl; NMR, nuclear
magnetic resonance; SP-HPLC, straight-phase high-performance liquid chromatography; UV,
ultraviolet.
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Abstract
The CYP74 family of cytochrome P450 enzymes consisting of allene oxide synthase, divinyl ether
synthase and hemiacetal synthase has important roles in plant oxylipin biosynthesis, transforming
lipoxygenase-generated fatty acid hydroperoxides into a range of biologically active compounds.
Biochemical and phylogenetic studies have shown that CYP74-related enzymes are found also in
marine invertebrates, such as coral, sea anemone and amphioxus (Branchiostoma floridae) (Lee, D.-S,
Nioche, P., Hamberg, M. and Raman, C.S., Nature 455:363-368 (2008)). Here, a CYP74 clan enzyme
from amphioxus, BfEAS, displaying epoxy alcohol synthase activity was incubated with
hydroperoxides of linoleic and linolenic acids and the products identified by chemical transformations,
mass spectrometry and NMR. Linoleic acid 13(S)-hydroperoxide was transformed into 12(R),13(S)-
epoxy-11(S)-hydroxy-9(Z)-octadecenoic acid, and an analogous conversion was observed with
linolenic acid 13(S)-hydroperoxide. Incubation of linoleic acid 13(S)-[18O2]-hydroperoxide led to the
formation of epoxy alcohol retaining both atoms of 18O, thus indicating a mechanism involving
homolysis of the hydroperoxide, formation of the epoxide by alkoxy radical attack at the α-
unsaturation, and rebound of hydroperoxide oxygen to form the 11(S) alcohol group. The 9(S)-
hydroperoxide of linoleic acid produced 9(S),10(R)-epoxy-11(S)-hydroxy-12(Z)-octadecenoic acid
accompanied by smaller amounts of 9(S),14(R,S)-dihydroxy-10(E),12(E)-octadecadienoic acid and the
new macrolactone 9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. In conclusion, three relevant
lipoxygenase hydroperoxides were converted to epoxy alcohols by BfEAS, supporting the discovery
that EAS constitutes a fourth category in the CYP74 clan of enzymes.
Supplementary Key Words Fatty acid hydroperoxide / CYP74 / Epoxy alcohol synthase /
amphioxus (Branchiostoma floridae)
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Introduction
Cytochrome P-450 enzymes belonging to the CYP74 family, i.e. allene oxide synthase (AOS),
divinyl ether synthase (DES) and hemiacetal synthase (HAS; also referred to as hydroperoxide lyase),
are responsible for most of the conversions which take place with lipoxygenase-generated
hydroperoxides in plants. Products formed in CYP74-initiated pathways include the jasmonates as
well as short-chain aldehydes and alcohols, compounds which have important roles to fulfill in plant
development and defense. Studies of the mechanism of CYP74-catalyzed reactions indicate that the
first step of hydroperoxide conversion results in an epoxyallylic radical which serves as a common
intermediate in the formation of products by AOS, DES and HAS (1). As seen in Fig. 1, the native
Fe(III) form of the various CYP74 enzymes is converted to Fe(IV)-OH during the initial step, and this
oxidized form is utilized to accomplish the subsequent transformations into allene oxides, divinyl
ethers and hemiacetals.
Epoxy alcohols constitute another group of biologically important oxylipins generated from
lipoxygenase-derived hydroperoxides. Two distinct mechanisms exist for the formation of epoxy
alcohols (2), i.e. either reduction of the hydroperoxide functional group into an alcohol and
epoxidation of one of the conjugated double bonds, or homolytic cleavage of the hydroperoxide O-O
bond, cyclization of the resulting alkoxy radical by attack at the neighboring unsaturated carbon atom
and recapture of OH. The former mechanism has been observed in the vanadium-promoted
degradation of hydroperoxides (3), for peroxygenase (4,5) and for a number of unidentified epoxy
alcohol synthases present in certain plants and fungi (ref. 6, and references cited therein). The latter
mechanism most often results in the formation of trans-configured epoxy alcohols and has been
observed for various nonenzymatic (7,8) and lipoxygenase-promoted conversions (9,10), as well as for
specific enzymatic hydroperoxide isomerizations catalyzed by lipoxygenase-type enzymes (11,12).
Recently structural, biochemical, and phylogenetic studies showed that amphioxus (Branchiostoma
floridae) contains several copies of CYP74-related genes, at least one of which encoding an epoxy
alcohol synthase (BfEAS) (13). The aim of the present work was to further characterize this enzyme
with respect to substrate specificity, products and mechanism.
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MATERIALS AND METHODS
Fatty acid hydroperoxides of >98% chemical purity were purchased from Lipidox, Stockholm,
Sweden. Erythro- and threo-10,11- and 11,12-dihydroxyoctadecanoates were prepared by cis-
hydroxylation (osmium tetroxide) or trans-hydroxylation (performic acid followed by saponification)
of the corresponding (Z)-monounsaturated fatty acid methyl esters (4). Standards of racemic and
optically active short chain 2-hydroxy acids used as references during steric analysis were available
since earlier work (cf. ref. 14,15). Agarose was purchased from Biozym Scientitic GmbH, and
restriction enzymes were from MBI Fermentas. Frozen adult amphioxus were a generous gift from Dr.
Linda Holland (Scripps Institution of Oceanography, University of California, San Diego, U.S.A.).
Cloning and expression
A codon-optimized gene encoding for BfEAS (CYP440A1, GenBank: ACD88492.1) (13) was
synthesized by the GENEWIZ Company (South Plainfield, USA) with a C-terminal hexahistidine
sequence essential for immobilized Ni-affinity chromatography. In order to improve expression of
soluble protein, the MAKKTSS-sequence was added at the N-terminus. The resulting construct was
cloned into the pET-vector and transformed into E. coli Bl21star. For heterologous expression the
recombinant cells were pre-cultivated in LB- or 2xYT-medium until an OD600 of approx. 0.6-0.8 was
reached. Protein expression was induced by the addition of 0.1 mM IPTG. 80 µg/mL δ-aminolevulinic
acid and 150 µM ammonium iron citrate were added at the time point of induction in order to ensure
that the co-factor availability is not limiting expression of BfEAS. After cultivation at 16 °C for 3 d
cells were harvested by centrifugation (8000 x g, 20 min, 4 °C), shock frozen in liquid nitrogen and
stored at -20 °C.
Cell lysis and affinity purification
Cell disruption was performed according to a protocol adapted from that established by
Richardson et al. (16). Briefly, cells from approx. 1L of culture were resuspended in 150 mL 100 mM
Tris/HCl (pH 7.8) containing 20 % glycerol. After addition of 0.2 mg/mL lysozyme the cell
suspension was stirringly incubated at 4 °C for 30 min and centrifuged for 10 min at 3000 x g.
Sedimented spheroblasts were dissolved in 50 mL of 10 mM phosphate buffer (pH 8.0) containing 14
mM MgAc2, 60 mM KAc, 0.1 mM DTT and were frozen at -80 °C (>12 h). PMSF was added to a
final concentration of 0.5 mM and cell lysis was increased by applying a sonifier cell disruptor B15
from Branson (5 x 1 min at 40% power and 40% pulse). Cell debris was removed by centrifugation for
20 min at 50 000 x g. The resulting supernatant was applied on a HisTrapTM HP column from GE
Healthcare that was pre-equilibrated with 50 mM phosphate buffer (pH 8.0) containing 1M NaCl and
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0.5 M urea. After washing with 10-20 column volumes of this buffer unspecifically bound proteins
were eluted by increasing the imidazol concentration to 15 mM. BfEAS was eluted by constantly
increasing the imidazol concentration to 0.3 M imidazol within 20 min at a flow rate of 1 mL/min. The
purity of BfEAS was assessed by means of SDS-PAGE analysis and Coomassie-staining.
Spectroscopic analysis
UV/vis spectra of the affinity purified BfEAS were performed at room temperature in phosphate
buffer (pH 8.0) using a Cary 100 Bio spectrophotometer from Varian. CD-spectra of BfEAS were
obtained under similar conditions using a Chirsacan CD Spetrometer from Applied Photophysics.
Incubations
The standard incubation of BfEAS, which was scaled up as needed, involved addition of
recombinant enzyme to the fatty acid hydroperoxide (300 µM) in 0.1 M potassium phosphate buffer
pH 7.4 (4 mL). The mixture was stirred at 23oC for 15 min and then extracted with diethyl ether. For
profiling of products using GC-MS, aliquots were treated with diazomethane followed by
trimethylchlorosilane-hexamethyldisilazane-pyridine (2:1:2, v/v/v) to generate methyl ester /
trimethylsilyl (TMS) ether derivatives.
For incubations with amphioxus homogenate, 150 mg of tissue in 10 mL of 0.1 M potassium
phosphate buffer pH 7.4 containing 100 µM linoleic acid was homogenized at 0o for 2 x 30 sec using
an Ultraturrax. The homogenate was stirred at 23o for 30 min, then extracted with diethyl ether.
Material was derivatized as methyl esters - TMS ether derivatives and analyzed by GC-MS using a
library of oxylipin standard mass spectra for product identification.
Preparative HPLC
Material generated in large scale incubations using 10-20 mg of hydroperoxide was treated with
diazomethane and subjected to straight-phase (SP) HPLC using a column of Nucleosil 50-7 (250 x 10
mm, Marcherey-Nagel, Düren, Germany). The column was eluted at a flow rate of 4 mL/min with
solvent systems of 0.3 - 5% 2-propanol in hexane as indicated. Serially connected detectors for
measurement of the absorption at 210 nm and refractive index were used.
Steric analysis
Analytes were derivatized to methyl ester / (-)-menthoxycarbonyl (MC) derivatives, purified by
TLC and subjected to oxidative ozonolysis as described (15). The methyl-esterified products
containing MC derivatives of short chain 2-hydroxyesters were analyzed by GC-MS using the
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corresponding derivatives of 2(S)- and 2(R,S)-hydroxyesters as references (14,15). For determination
of erythro-/threo configurations of vicinal methyl dihydroxyoctadecanoates, samples were derivatized
to bis-TMS ether derivatives and the retention times on GC-MS were recorded.
GC-MS analysis
A Hewlett-Packard model 5970B mass selective detector connected to a Hewlett-Packard model
5890 gas chromatograph equipped with a 12 m phenylmethylsilicone capillary column was used for
GC-MS. Helium was used as the carrier gas. The oven temperature was raised from 120oC to 300oC at
a rate of 10oC/min. Alternatively, GC-MS analyses were performed using a Shimadzu QP5050A mass
spectrometer connected to Shimadzu GC-17A gas chromatograph equipped with an MDN-5S (5%
phenyl 95% methylpolysiloxane) fused capillary column (length, 30 m; ID 0.25 mm; film thickness,
0.25 µm). Helium at a flow rate of 30 cm/s was used as the carrier gas. Injections were made in the
split mode using an initial column temperature of 120 °C. The temperature was raised at 10 °C/min
until 240 °C. Full scan or selected ion monitoring (SIM) analyses were both performed using electron
impact ionization at 70 eV.
NMR spectroscopy
The 1H-NMR, COSY, NOESY, HSQC, HSQC-TOCSY and HMBC spectra (600 MHz,
[2H6]benzene, 296 K) were recorded with Bruker Avance III 600 instrument.
RESULTS
Expression and purification
The BfEAS-gene used in the present study has previously been identified and partly
characterized by Lee and co-workers (13). We adapted the reported protocol by using the pET-vector
system for recombinant expression in analogy to the method recently established for the analysis of
CYP74-enzymes from the moss Physcomitrella patens (17). Briefly, we used a cloning strategy that
led to the expression of BfEAS with a C-terminal hexahistidine-tag and an N-terminal MAKKTSS-
sequence extension. Expression of soluble protein was improved by the addition of iron ammonium
citrate and δ-amino-levulinic acid at the time point of induction. Using a single affinity purification
step we were able to obtain BfEAS with a purity of >90% (Fig. 2).
Spectral properties
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We observed a reddish/brownish color of the protein fraction eluted from the immobilized Ni2+-
column indicating the presence of heme as a co-factor. Indeed, when we analyzed the UV/VIS
spectrum of BfEAS we found an absorption profile characteristic for heme proteins. Four distinct
absorption maxima were observed at 565 nm (α), 539 nm (β), 420 nm (γ, Soret-band), and 368 nm (δ)
(Fig. 3). The relatively low absorption of the Soret-band indicated that the heme-occupancy was
significantly reduced as it has previously been observed for recombinant CYP74-enzymes from P.
patens (17). We calculated the approximate heme content of recombinant BfEAS as 37% based on a
theoretical molar extinction coefficient at 280nm (ε280nm = 89 500 1/(M.cm)) and an expected molar
extinction coefficient of the Soret-band of εSoret = 100 000 1/(M.cm). In order to gather information
about the secondary structure of BfEAS we recorded CD-spectra of the purified enzyme in the far UV.
The respective spectrum is shown in Fig. 3. It shows to main negative peaks at 212 nm and 222 nm
and positive peak at 194 nm. These signals are indicative for proteins with a mainly alpha-helical fold.
Structures of reaction products formed from fatty acid hydroperoxides incubated with BfEAS
Compound 1 – A GC-MS chromatogram of products (Me ester/TMS derivatives) formed upon
incubation of 13(S)-HPODE with BfEAS is shown in Fig. 4A. Compound 1 constituted >95% of the
products (the structural formulae of Compound 1 and other products are shown in Fig. 5). The mass
spectrum (Me/TMS, Fig. 4B) showed [M – Me]+ at m/z 383 and a prominent fragment TMSO+=CH-
CH=CH-(CH2)7-COOCH3 at m/z 285. These results suggested the structure 12,13-epoxy-11-hydroxy-
9-octadecenoic acid as shown in the fragmentation scheme (Fig. 4B, insert).
Larger amounts of the methyl ester of Compound 1 required for structural analysis was obtained
following purification of methyl-esterified material from pooled standard incubations by SP-HPLC
using a solvent system of 2.5% 2-propanol in hexane.
The NMR data for Compound 1 (Me ester) are presented in Table 1. The double bond has the
"Z" configuration as seen from the J9,10 = 11 Hz, and the epoxide is cis-configured as shown by the
J12,13 = 4.3 Hz. The spin-spin interaction between H11 and H12 (J11,12 = 7.7 Hz) does not allow
assignment of the erythro/threo configuration because of the small differences between the relevant
coupling constants in the NMR spectra of erythro and threo cis-epoxyalcohols (12). However, the 2D-
NOESY spectrum (data not shown) revealed an intense cross-peak between H11 and H12 as well as a
less prominent cross-peak between H11 and H13, and these nuclear Overhauser effects indicate the
spatial proximity between these protons (especially H11 and H12) in agreement with the threo
configuration. In addition, the 2D-NOESY data showing a pronounced cross-peak between H11 and
H14a,b gave further evidence for the presence of a cis-epoxide group.
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An aliquot of 100 µg of the methyl ester of Compound 1 was hydrogenated with 5 mg of Pt
catalyst in 1 mL of methanol affording the dihydro derivative. The epoxide function was
deoxygenated by treatment with triphenylphosphine selenide (31 mg) and TFA (3.5 mg) in 1 mL of
CH2Cl2 (14). The methyl 11-hydroxy-12-octadecenoate resulting from this treatment (Fig. 6) was
converted to the MC derivative and subjected to oxidative ozonolysis (14,15). Analysis of the methyl-
esterified product by GC-MS showed the formation of the MC derivative of dimethyl 2(S)-
hydroxydodecane-1,12-dioate, thus demonstrating that Compound 1 had the “S” configuration at C-11.
Another part of the methyl ester of Compound 1 (100 µg) was hydrogenated with 5 mg of palladium-
on-carbon in 1 mL of methanol affording methyl 11,12-dihydroxyoctadecanoate as the main product
(Fig. 6). The structure of this hydrogenolysis product was demonstrated by the mass spectrum
recorded on the TMS derivative showing prominent ions at m/z 443 (M+ - 31; loss of OCH3), 360 (M+
- 114; loss of OHC-(CH2)5-CH3), 287 (TMSO+=CH-(CH2)9-COOCH3), and 187 (TMSO+=CH-(CH2)5-
CH3). GC-MS analysis using standards of the corresponding derivatives of erythro- and threo-11,12-
dihydroxyoctadecanoic acids (retention times, 12.98 min (erythro) and 12.86 min (threo)) showed that
the BfEAS-derived material cochromatographed with the latter of these isomers, thus establishing the
threo relative configuration between the C-11/C-12 oxygens in Compound 1.
In conclusion, Compound 1 formed from 13(S)-HPODE in the presence of BfEAS was
identified as the cis-epoxy alcohol 12(R),13(S)-epoxy-11(S)-hydroxy-9(Z)-octadecenoic acid.
Compound 2 – GC-MS analysis of products (Me/TMS) formed upon incubation of 13(S)-
HPOTrE with BfEAS revealed a single predominant product, i.e. Compound 2 (Fig. 4D). The mass
spectrum (not illustrated) showed M+ at m/z 396 (0.02%), [M – Me]+ at m/z 381 (0.3%), and
TMSO+=CH-CH=CH-(CH2)7-COOCH3 at m/z 285 (73%). These data enabled us to ascribe the
structure of 12,13-epoxy-11-hydroxy-9,15-octadecadienoic acid to Compound 2, however the full
stereochemistry was not determined. Two minor products (< 1% of the product) were identified as the
α-ketol 13-hydroxy-12-oxo-9,15-ocatecadienoic acid and the cyclopentenone cis-12-oxo-10,15-
phytodienoic acid. Such AOS products were not detected after incubation of 13(S)-HPODE with
BfEAS.
Compound 4 – Three products appeared following incubation of 9(S)-HPODE with BfEAS as
determined by GC-MS analysis, i.e. Compounds 3, 4 and 5 (Fig. 7A); of these, Compound 4 was the
major product. The methyl ester derivatives of Compound 4 and 5 were obtained in pure form
following SP-HPLC using solvent systems of 2.5% 2-propanol in hexane (methyl ester of Compound
4) and 5% 2-propanol in hexane (methyl ester of Compound 5, two diastereomers).
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The mass spectrum (Fig. 7C) of Compound 4 (Me ester - TMS derivative) showed M+ at m/z
398 (0.1%); [M – Me]+ at m/z 383 (1%); [M – OHC(CH2)7COOCH3]+ at m/z 212 (3%) and
TMSO+=CH-CH=CH-(CH2)4-CH3 at m/z 199 (100%), as shown in the fragmentation scheme (Fig. 7C,
insert). The NMR spectral parameters of Compound 4 Me ester (suppl. Table 1) were nearly identical
to those of Compound 1. The spin-spin coupling value J12,13 = 11.0 Hz indicated that the double bond
had the "Z" configuration. The oxirane protons had cis assignment as seen from their coupling
constant value J9,10 = 4.3 Hz. The spin-spin coupling constant of the secondary alcohol methine (C11)
and the neighboring oxirane methine (C10), J10,11 = 7.7 Hz, did not provide information about the
erythro/threo relationship as mentioned above. However, the 2D-NOESY data exhibited a nuclear
Overhauser effect, a pronounced cross-peak between H10 and H11, which was in agreement with the
threo configuration.
An aliquot of the methyl ester of Compound 4 (100 µg) was subjected to catalytic
hydrogenation using Pt catalyst followed by deoxygenation as described above for the methyl ester of
Compound 1; this afforded methyl 11-hydroxy-9-octadecenoate. Derivatization of the material to its
MC derivative and oxidative ozonolysis generated the MC derivative of 2(S)-hydroxynonanoic acid
having a stereochemical purity of about 92%, thus demonstrating that the absolute configuration at C-
11 of Compound 4 was mainly "S". In another experiment, 100 µg of the methyl ester of Compound 4
was hydrogenated using palladium-on-carbon. The TMS derivative of the resulting hydrogenolysis
product, i.e. methyl 10,11-dihydroxyoctadecanoate, was analyzed by GC-MS using references of the
authentic erythro- and threo-10,11-diols (threo diol eluting 0.12 min ahead of the erythro diol). This
analysis showed that the 10,11-dihydroxyoctadecanoate derived from Compound 4 was mainly due to
the threo isomer, i.e. threo/erythro isomers appearing in a ratio of 93:7.
These results, demonstrated that Compound 4 was mainly due to 9(S),10(R)-epoxy-11(S)-
hydroxy-12(Z)-octadecenoic acid, although 7-8% appeared to exist with the 11(R) configuration.
Compound 5 – The mass spectrum of Compound 5 (Me/TMS) is shown in Fig. 7D. The
spectrum possessed M+ at m/z 470 (0.5%); [M – Me]+ at m/z 455 (0.4%); [M – TMSOH]+ at m/z 380
(22%), as well as other fragment ions depicted in the fragmentation scheme (Fig. 7D, insert). These
mass spectral data suggested the presence of a 9,14-diol structure.
The methyl ester of Compound 5 isolated as described above was subjected to NMR analyses.
The NMR data (suppl. Table 2), including the 1H-NMR, 2D-COSY, NOESY, HSQC and HMBC)
demonstrated the structure 9,14-dihydroxy-10(E),12(E)-octadecadienoic acid (Me ester). The signals
of protons H9/H14, H10/H13 and H11/H12 exhibited a pairwise coincidence due to the symmetrical
structure of two conjugated double bonds placed between two secondary alcohol functions. A complex
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multiplicity of signals of double bond methins in a 1H-NMR spectrum does not allow estimating the
spin-spin coupling constant values. However, the data of HSQC-TOCSY enabled us to measure the
spin constants J10,11 = J12,13 = 16.2 Hz. This showed that the double bonds at C10 and C12 had both the
"E" configuration.
Steric analysis of the methyl ester - MC derivative of Compound 5 (100 µg) showed the
formation of the MC derivatives of dimethyl 2(S)-hydroxydecane-1,10-dioate and methyl 2(R,S)-
hydroxyhexanoate, thus proving the presence in Compound 5 of a 9(S)-hydroxyl group, a racemic
alcohol function at C-14, and a conjugated diene structure located at C-10 - C-13. Together with the
NMR data, these results identified Compound 5 as 9(S),14(R,S)-dihydroxy-10(E),12(E)-
octadecadienoic acid.
Compound 3 – Compound 3 appeared as a minor component constituting about 3% of the
products formed upon incubation of 9(S)-HPODE with BfEAS (Fig. 7A). In contrast to all other
products formed by BfEAS, analysis of Compound 3 by GC-MS did not require methyl-esterification
indicating that the carboxyl group was derivatized. It was also the least polar of the products
encountered, and a convenient way to generate large amounts for structural work was to perform two-
phase incubations in the following way. 9(S)-HPODE (320 µM) was added to a mixture of 40 mL of
potassium phosphate buffer pH 7.4 containing BfEAS and 40 mL of hexane. The two-phase system
was vigorously shaken for 15 min and the hexane layer was taken to dryness. Analysis of the material
by GC-MS (before and after treatment with diazomethane and trimethylsilylating reagent) showed the
presence of Compound 3 as the sole product. Material for structural work was obtained by performing
several such incubations using totally 14 mg of 9(S)-HPODE followed by purification by SP-HPLC
with 0.3% 2-propanol in hexane as the solvent system; this afforded 2.1 mg of Compound 3 as a
colorless oil.
The mass spectrum of Compound 3 (Fig. 7B) showed M+ at m/z 294 (0.6%), [M – CH3(CH2)4]+
at m/z 223 (6%), [M – CH3(CH2)4CHCH=CHCH]+ at m/z 171 (13%), and [M –
CH3(CH2)4CHCH=CHCHO]+ at m/z 155 (49%). The proposed structure and its mass fragmentation
scheme is shown in Fig. 7B (insert). For further elucidation of the structure of Compound 3 we
recorded its NMR spectra.
The NMR spectrum of Compound 3 (Fig. 8 and suppl. Table 3) possessed some features
atypical for ordinary straight chain oxylipins. The heteronuclear multiple bond correlations revealed
the neighborhood of C1 and C13, thus indicating the existence of an intramolecular ester bond. The
H13 signal was strongly shifted downfield to 5.51 ppm due to the deshielding effect of the
neighboring carbonyl (C1), also confirming the ester link between C1 and C13. All methylene protons
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from H2 to H8 are chemically non-equivalent since they belong to a macrolactone ring. The oxirane
protons had a coupling constant J9,10 = 4.2 Hz, thus proving a cis-epoxide. The olefinic protons
exhibited J11,12 = 15.4 Hz, demonstrating that the double bond had the "E" configuration. Taken
together, the MS and NMR data enabled us to ascribe the structure cis-9,10-epoxy-11(E)-octadecen-
13-olide to Compound 3.
The stereochemical features of Compound 3 were determined by the conversions shown in Fig.
9. Compound 3 (500 µg) was dissolved in 0.5 mL of methanol and 4.5 mL of water and 50 µL of 2 M
HCl were added. After stirring for 10 min at 23oC, material was extracted with diethyl ether. An
aliquot was analyzed GC-MS as the TMS derivative showing two peaks of isomers in a 3:1 ratio. The
deduced molecular weight of this material was 312, corresponding to addition of one H2O to
Compound 3 (Fig. 9). Subsequent treatment with 0.2 M NaOH in 90% aqueous methanol at 23oC for
18 h liberated the carboxyl in its free form and afforded two major trihydroxy-octadecenoate isomers.
The methyl ester - TMS derivatives of those showed the following prominent mass spectral ions: m/z
545 (M+ - 15; loss of CH3), 460 (M+ - 100; rearrangement and loss of OHC-(CH2)4-CH3), 259 (100%;
TMSO+=CH-(CH2)7-COOCH3), and 155 (isomer a), and m/z 460 (M+ - 100; rearrangement and loss of
OHC-(CH2)4-CH3), 259 (TMSO+=CH-(CH2)7-COOCH3), and 173 (100%; TMSO+=CH-(CH2)4-CH3)
(isomer b) (Fig. 9). Using mass spectral ions and GLC retention times, the two isomers could be
structurally and stereochemically matched with isomeric trihydroxyoctadecenoates known since
previous work (15); this demonstrated that isomer a was identical to methyl 9(S),10(S),13(S)-
trihydroxy-11(E)-octadecenoate whereas isomer b was identical to methyl 9(S),12(R),13(S)-
trihydroxy-10(E)-octadecenoate. Independent support for the configurations at C-13 and C-9 of
isomers a and b was provided by oxidative ozonolysis which produced 2(S)-hydroxyheptanoic acid
and 2(S)-hydroxydecane-1,10-dioic acid as the main chiral fragments. On the basis of these findings
and the NMR data, it was concluded that Compound 3 had the structure 9(S),10(R)-epoxy-11(E)-
octadecen-13(S)-olide.
18O Labeling Experiments
In order to study the origin of the epoxy and hydroxy oxygen atoms in Compound 1 we
performed incubations of BfEAS with [18O2-hydroperoxy]13(S)-HPODE, and of unlabeled 13(S)-
HPODE in [18O]H2O. The products (Me/TMS) were analyzed by GC-MS. The mass spectrum of
derivatized Compound 1 biosynthesized from unlabelled 13-HPODE showed M+ at m/z 398 and [M –
Me]+ at m/z 383 (Fig. 4B). In contrast, Compound 1 (Me/TMS) formed upon BfEAS incubation with
[18O2]13-HPODE exhibited M+ at m/z 402 and [M – Me]+ at m/z 387 (Fig. 4C, Table 2), thus
indicating the incorporation of both 18O atoms from the hydroperoxide into the product. Selected ion
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monitoring (SIM) analyses of products (Me/TMS) and integration of the intensity of ions at m/z 402.4,
400.4 and 398.4 enabled us to quantify the molecular species of Compound 1 having 2, 1 and 0
incorporated 18O atoms, respectively (Table 2A). The species containing two 18O atoms constituted
94.93% of total integral intensities of three species. The estimated total isotopic content of 18O in
Compound 1 was 96.12%, while the [18O2]13-HPODE precursor contained 96.98% of 18O (Table 2B).
Thus, the experiments revealed nearly quantitative incorporation of two 18O atoms from [18O2]13-
HPODE into Compound 1. Upon incubation of BfEAS with unlabeled 13-HPODE in [18O]water (98% 18O) no incorporation of 18O into Compound 1 was observed as shown by SIM GC-MS.
Identification of products formed from linoleic acid incubated with amphioxus homogenate
Analysis of the derivatized reaction product formed from linoleic acid incubated with
amphioxus whole homogenate showed the presence of three major compounds, i.e. 9-hydroxy-10,12-
octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid and 9,10-epoxy-13-hydroxy-11-
octadecenoic acid. Smaller amounts of 9-keto-10,12-octadecadienoic acid, isomeric 9,10,13- and
9,12,13-trihydroxyoctadecenoates, and 2-hydroxylinoleic acid were also observed. Because of the
limited amounts of material available, the stereochemistry of these products was not determined.
Linoleic acid is an endogenous fatty acid in amphioxus (18), and its metabolism by lipoxygenase
apparently involves 9-LOX activity.
DISCUSSION
The bond dissociation energy of the oxygen-oxygen bond of the hydroperoxy group is only
about 44 kcal/mol, and the facile homolytic cleavage of this bond accounts for most of the reactivity of
fatty acid hydroperoxides. The resulting alkoxy radical can undergo a number of transformations, one
of which consists of intramolecular attack at the neighboring unsaturated carbon atom forming a
delocalized epoxyallylic radical (7,8). Already 40 years ago structures produced by trapping of such
radicals were published, i.e. formation of epoxy adducts to tocopherol (19) as well as a specific epoxy
alcohol during heating of a fatty acid hydroperoxide (20). Since then, numerous agents have been
found to promote epoxy alcohol formation by the homolytic hydroperoxide cleavage/cyclization route,
e.g. ferrous ion and other metal ions, hemin, hemoglobin, and UV-light (7,8). Soybean lipoxygenase
operating under anaerobic conditions also produces epoxy alcohols together with other products (9).
Hepoxilins constitute a group of eicosanoids formed from arachidonic acid 12-hydroperoxide in the
presence of e.g. 12-lipoxygenase (10), and a specific conversion of arachidonic acid 12(R)-
hydroperoxide into the epoxy alcohol 11(R),12(R)-epoxy-8(R)-hydroxy-5(Z),9(E),14(Z)-eicosatrienoic
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acid is catalyzed by the hydroperoxide isomerase activity of epidermal lipoxygenase type 3 (eLOX3)
(11). In a recently published study, the cis-configured epoxy alcohol 8(R),9(S)-epoxy-10(S)-hydroxy-
11(Z),14(Z)-eicosadienoic acid was identified as the product formed from dihomo-γ-linolenic acid
incubated with an 8(R)-lipoxygenase from the coral Plexaura homomalla (12).
Earlier studies have shown that P450s (21) and AOS (22) can produce epoxy alcohols in
reactions which are considerably more specific compared to simple hematin-promoted hydroperoxide
degradations, however, a CYP74 clan enzyme acting as a true EAS was reported only recently (13). In
that study a recombinant CYP74 clan enzyme from amphioxus produced >95% of a cis-configured
epoxy alcohol when incubated with 13(S)-HPODE. The present paper reports further studies of this
enzyme regarding substrates, products and mechanism.
The hydroperoxides 13(S)-HPODE and 13(S)-HPOTrE were each converted into a single epoxy
alcohol by BfEAS, i.e. 12(R),13(S)-epoxy-11(S)-hydroxy-9(Z)-octadecenoic acid (Compound 1) and
12,13-epoxy-11-hydroxy-9,15-octadecadienoic acid (Compound 2), respectively. Studies with 13(S)-
[18O2]HPODE revealed that both hydroperoxide oxygens were fully retained in the epoxy alcohol
product, thus excluding mechanisms involving an epoxyallylic carbocation and incorporation of OH-
from water (23). The 9-LOX-derived hydroperoxide 9(S)-HPODE also produced an epoxy alcohol as
the main product, i.e. 9(S),10(R)-epoxy-11(S)-hydroxy-12(Z)-octadececenoic acid (Compound 4). The
general mechanism postulated for formation of epoxy alcohols by BfEAS (Fig. 10) involves initial
formation of an alkoxy radical by hydroperoxide O-O homolysis followed by cyclization into an
epoxyallylic radical and formation of the C-11 hydroxyl group by rebound of hydroperoxide oxygen
via Fe(IV)-OH. Two additional products formed from 9(S)-HPODE upon incubation with BfEAS
were characterized, i.e. Compound 5, which was identified as the diol 9(S),14(R,S)-dihydroxy-
10(E),12(E)-octadecadienoic acid, and Compound 3, which was identified as the macrolactone
9(S),10(R)-epoxy-11(E)-octadecen-13(S)-olide. It seems likely that formation of these products took
place from a common epoxyallylic carbocation intermediate formed by rearrangement and 1-electron-
oxidation of the epoxyallylic radical (Fig. 10). Loss of a proton from C-14 would lead to an
epoxydiene, which according to previous work (24,25) is quite unstable and suffers spontaneous
hydrolysis by solvent attack at C-14, thus forming the diol isolated. Alternatively, attack by the
carboxylate group at the C-13 carbocation will lead to formation of the macrolactone product.
Somewhat unexpectedly, this attack occurred in a stereospecific way, most likely when the
carbocation was still bound to the enzyme, and provided only the 13(S)-configured product. Another
example of stereospecific formation of an oxylipin macrolactone was reported very recently (26).
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Whereas the AOS and DES enzymes of the CYP74 family catalyze a formal dehydration of
their substrates, the EAS described in the present paper acts as a hydroperoxide isomerase. One more
member of the CYP74 family, i.e. HAS (also referred to as hydroperoxide lyase; belonging to the
CYP74B and CYP74C subgroups), has been shown to be a hydroperoxide isomerase, in this case
catalyzing formation of unstable fatty acid hemiacetals as shown in Fig. 1. Experiments with 18O2-
labeled hydroperoxides have revealed that both 18O atoms from hydroperoxides are incorporated into
hemiacetals (27-29); therefore the hydroperoxide homolysis/rebound mechanism is common to EAS
and HAS.
The possible function in amphioxus of the linoleic acid-derived CYP74 products encountered in
the present study is entirely unknown. When homogenates of amphioxus were incubated with linoleic
acid, exclusive formation of 9-LOX products were observed, therefore suggesting that Compounds 3-5
may be biologically relevant in amphioxus. However, linoleic acid is a minor fatty acid in amphioxus
(18), and it is possible that analogous or related products formed from the major amphioxus fatty acid,
i.e. docosahexaenoic acid (18), are of greater biological importance.
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ACKNOWLEDGMENTS
This work was supported by grant 12-04-01140-a from the Russian Foundation for Basic Research, a
grant from the Russian Academy of Sciences (program 'Molecular and Cell Biology'), a grant from the
program "Leading Scientific Schools", grant 14.740.11.0797 from the Federal Target Program (A.N.
Grechkin and B.I. Khairutdinov), and a grant from the Deutsche Forschungsgemeinschaft to Ivo
Feussner in the framework of the International Research Training Group (IRTG) 1422. Julia Scholz
was additionally supported by the Biomolecules program of the Göttingen Graduate School of
Neurosciences and Molecular Biology (GGNB).
The authors thank Gunvor Hamberg, Pia Meyer and Sabine Freitag for their skillful technical
assistance and Drs. Fahima Mukhitova and Anna Ogorodnikova for their help at separate stages of the
work.
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TABLE 1. NMR spectral data for Compound 1 Me ester
Position number
13C chemical shifts (ppm); functional group
1H chemical shifts (ppm); multiplicity; coupling constant (Hz)
Heteronuclear multiple bond correlation
1 173.53; COOMe COOMe, H2, H3
2 34.24; CH2 2.11; t; 7.4 (H3) C3, C4, COO, COOMe, H3
3 25.36; CH2 1.54; m C2, C4, COO, COOMe, H2
4 29.51; CH2 1.15; m C2, C3, H2, H3
5 28.38-30.70; CH2 1.07-1.31; m
6 29.38; CH2 1.14; m H8a, H8b
7 30.01; CH2 1.21; m C9, H8a, H8b, H9
8a 28.48; CH2 1.92; m C6, C7, C9, H8b, H9
8b 2.01; m C7, C9,C10, H7, H8a
9 134.03; CH 5.44; dt; 11.0 (H10); 6.9 (H8a,b)
C7, C8, C10, H8a, H8b,
10 128.46; CH 5.49; ddt; 8.5 (H11); 1.2 (H8a,b)
C8, C9, C11, C12, H8a, H8b, H9, H11
11 66.77; CH 4.28; dd; 7.7 (H12) C10, C12, C13, H10, H12, OH,
12 60.74; CH 2.91; dd; 4.3 (H13) C10, C11, c13, C14, H10, H11, H13, H14
13 57.82; CH 2.74; ddd; 7.3 (H14a); 4.9 (H14b)
C14, C15, H11, H12, H13, H14, H15
14 29.09; CH2 1.42; m, AB C12, C13, C15, C16, H12, H13, H15a
15a 27.08; CH2 1.28; m C13, C14, C16, H13, H14, H17
15b 1.38; m C13, C14, C16, H13, H14, H17
16 32.16; CH2 1.23; m C16, C18, H14, H15, H18
17 23.08; CH2 1.21; m C15, C16, H15a, H15b, H18
18 14.39; CH3 0.87; t; 7.1 (H17) C16, C17, H16, H17
(1) 51.14; COOMe 3.37; s C2, COO, H2
(11) OH 1.80; d H11
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TABLE 2. 18O incorporation from [18O2-hydroperoxy]13(S)-HPODE into Compound 1 in the presence of BfEAS
A. GC-MS (SIM) quantification of Compound 1 species possessing 2, 1 or 0 atoms of 18O (M+ at m/z 402.4, 400.4 and 398.4, respectively).
Ion, m/z Integral intensities of molecular ion M+ peaks in SIM GC-MS chromatograms of 18O labelled and unlabelled Compound 1
BfEAS + [18O2-hydroperoxy]13-HPODE
BfEAS + unlabelled 13-HPODE
398.4 2.64±0.38 93.13±0.88
400.4 2.37±0.19* 6.72±0.56
402.4 94.93±0.56* 0.30±0.03
*Presented relative intensities of ions (m/z 400.4 and 402.4) in spectrum of 18O labelled product were corrected in accordance with the relative abundance of the same ions in the spectrum of unlabelled product.
B. The total isotopic content of 18O in Compound 1 and the precursor [18O2]13-HPOD
Total estimated 18O content in product 1 (100% corresponds to two 18O atoms incorporated)
Control (18O content in [18O2-hydroperoxy]13-HPODE)
96.12±0.58 96.98 ± 0.22%**
**[18O2]13-HPODE was successively reduced with sodium borohydride, methylated with diazomethane and trimethylsilylated. The resulting [18O]13-HODE was subjected to SIM GC-MS analyses. 18O content was estimated from the relative abundance of ion pairs 384.4 and 382.4, [M]+; 313.3 and 311.3 [M-C5H11]+; 227.2 and 225.2 [M-(CH2)8COOMe]+.
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Figure legends
Figure 1: General mechanisms in the formation of CYP74 products illustrated with the
hydroperoxide 13(S)-HPODE as substrate. AOS, allene oxide synthase; DES, divinyl ether
synthase; HAS, hemiacetal synthase (earlier referred to as hydroperoxide lyase). The allene oxide and
hemiacetal products are both unstable and spontaneously converted to ketols and short chain
aldehydes, respectively.
Figure 2: SDS-PAGE analysis of BfEAS purification. BfEAS was heterologously expressed in E.
coli Bl21 star at 16°C for 3d and harvested by centrifugation. After cell disruption via sonification the
cell free extract was applied on a Ni-NTA column. Protein elution was performed with a linear
gradient of increasing imidazol concentration. Shown are the following fractions: unbound protein
(FT), washed proteins (W1, W2) and eluted proteins.
Figure 3: Spectroscopic analysis of recombinant BfEAS. Shown is a typical UV/vis-spectrum
(upper) and a CD-spectrum (lower) of purified BfEAS in sodium phosphate buffer (50 mM, pH 8.0).
The UV/vis-spectrum shows two major absorption maxima at 281 nm and 420 nm (γ, soret-band)
typical for heme proteins. Additionally three smaller maxima are present at 368 nm (δ), 539 nm (β)
and 565 nm (α). The CD-spectrum shows major minima at approx. 215 nm and 225 nm that are
characteristic for α-helical proteins.
Figure 4: GC-MS analyses of products (Me/TMS) formed from 13-hydroperoxides incubated
with BfEAS. A. GC-MS chromatogram of 13(S)-HPODE products. B. Mass spectrum of Compound
1; insert: fragmentation scheme. C. Mass spectrum of Compound 1 biosynthesized from [18O2-
hydroperoxy]13(S)-HPODE; insert: fragmentation scheme. D. GC-MS chromatogram of 13(S)-
HPOTrE products. "1", 12,13-epoxy-11-hydroxy-9-octadecenoic acid; "2", 12,13-epoxy-11-hydroxy-
9,15-octadecadienoic acid; "12-oxo-PDA", cis-12-oxo-10,15-phytodienoic acid; "α-ketol", 13-
hydroxy-12-oxo-9,15-octadecadienoic acid.
Figure 5: Structures of BfEAS products.
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Figure 6: Chemical transformations carried out on Compound 1. TPP=Se, triphenylphosphine
selenide; TFA, trifluoroacetic acid; MCCl, (-)-menthoxycarbonyl chloride.
Figure 7: GC-MS analyses of products (Me/TMS) formed from 9(S)-HPODE incubated with
BfEAS. A. GC-MS chromatogram of 9(S)-HPODE products. B. Mass spectrum of Compound 3;
insert: fragmentation scheme. C. Mass spectrum of Compound 4; insert: fragmentation scheme. D.
Mass spectrum of Compound 5; insert: fragmentation scheme. "3", 9,10-epoxy-11-octadecen-13-olide;
"4", 9,10-epoxy-11-hydroxy-12-octadecenoic acid; "5", 9,14-dihydroxy-10,12-octadecadienoic acid.
Figure 8: NMR data for Compound 3 produced from 9(S)-HPODE upon incubation with
BfEAS. A. 2D-COSY spectrum. Top and left projections: 1H-NMR spectrum. Insert: structure of
Compound 3 with specified carbon chain numbering. B. 1H-13C-HSQC spectrum. Left projection: 13C-
NMR spectrum.
Figure 9. Chemical transformations carried out on Compound 3. The trihydroxyoctadecenoates
"isomer a" and "isomer b" were assigned the 9(S),10(S),13(S) and 9(S),12(R),13(S) absolute
configurations, respectively, by GC-MS analysis using authentic standards as described in ref. 15.
Figure 10. Reactions and mechanisms in the formation of products catalyzed by BfEAS. 13(S)-
HPODE (precursor of Compound 1): R = -(CH2)4CH3, R’ = -(CH2)6COOH; 13(S)-HPOTrE (precursor
of Compound 2): R = -CH2CH=CH-CH2CH3; R’ = -(CH2)6COOH; 9(S)-HPODE (precursor of
Compounds 4, 5 and 3): R = -(CH2)7COOH; R’ = -(CH2)3CH3.
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Figure 2.
18 mM 240 mM
kDa 116.0
66.2
45.0
◄ BfEAS
35.0
FT W1 W2 MW
Elution (increasing imidazol conc.)
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Figure 3.
190 200 210 220 230 240 250 260
-6
-4
-2
0
2
4
6
8
CD [m
deg]
wavelength [nm]
300 400 500 6000.0
0.2
0.4
0.6
0.8
1.0ab
sorp
tion
wavelength [nm]
281 nm
420 nm
539 nm 565 nm
368 nm
wavelength [nm]
abso
rptio
nCD
[mde
g]
wavelength [nm]
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