Oxidation of C18 Hydroxy-Polyunsaturated Fatty Acids to Epoxide or Ketone by Catalase-Related Hemoproteins Activated with Iodosylbenzene

Article abstract of DOI:10.1007/s11745-017-4271-0

Small catalase-related hemoproteins with a facility to react with fatty acid hydroperoxides were examined for their potential mono-oxygenase activity when activated using iodosylbenzene. The proteins tested were a Fusarium graminearum 41?kD catalase hemoprotein (Fg-cat, gene FGSG_02217), a Pseudomonas fluorescens Pfl01 catalase (37.5?kD, accession number WP_011333788.1), and a Mycobacterium avium ssp. paratuberculosis 33?kD catalase (gene MAP-2744c). 13-Hydroxy-octadecenoic acids (which are normally unreactive) were selected as substrates because these enzymes react specifically with the corresponding 13S-hydroperoxides (Pakhomova et al. 18:2559–2568, 5; Teder et al. 1862:706–715, 14). In the presence of iodosylbenzene Fg-cat converted 13S-hydroxy-fatty acids to two products: the 15,16-double bond of 13S-hydroxy α-linolenic acid was oxidized stereospecifically to the 15S,16R-cis-epoxide or the 13-hydroxyl was oxidized to the 13-ketone. Products were identified by UV, HPLC, LC–MS, NMR and by comparison with authentic standards prepared for this study. The Pfl01-cat displayed similar activity. MAP-2744c oxidized 13S-hydroxy-linoleic acid to the 13-ketone, and epoxidized the double bonds to form the 9,10-epoxy-13-hydroxy, 11,12-epoxy-13-hydroxy, and 9,10-epoxy-13-keto derivatives; equivalent transformations occurred with 9S-hydroxy-linoleic acid as substrate. In parallel incubations in the presence of iodosylbenzene, human catalase displayed no activity towards 13S-hydroxy-linoleic acid, as expected from the highly restricted access to its active site. The results indicated that with suitable transformation to Compound I, monooxygenase activity can be demonstrated by these catalase-related hemoproteins with tyrosine as the proximal heme ligand.

Full text of DOI:10.1007/s11745-017-4271-0

Lipids (2017) 52:587–597
DOI 10.1007/s11745-017-4271-0
ORIGINAL ARTICLE
Oxidation of C18 Hydroxy‑Polyunsaturated Fatty Acids
to Epoxide or Ketone by Catalase‑Related Hemoproteins
Activated with Iodosylbenzene
Tarvi Teder1,2 · William E. Boeglin1 · Alan R. Brash1
Received: 25 April 2017 / Accepted: 2 June 2017 / Published online: 19 June 2017
© AOCS 2017
Abstract Small catalase-related hemoproteins with a
facility to react with fatty acid hydroperoxides were
examined for their potential mono-oxygenase activity
when activated using iodosylbenzene. The proteins tested
were a Fusarium graminearum 41 kD catalase hemo-
protein (Fg-cat, gene FGSG_02217), a Pseudomonas
fuorescens Pf01 catalase (37.5 kD, accession number
WP_011333788.1), and a Mycobacterium avium ssp.
paratuberculosis 33 kD catalase (gene MAP-2744c).
13-Hydroxy-octadecenoic acids (which are normally
unreactive) were selected as substrates because these
enzymes react specifcally with the corresponding
13S-hydroperoxides (Pakhomova et al. 18:2559–2568, 5;
Teder et al. 1862:706–715, 14). In the presence of iodo-
sylbenzene Fg-cat converted 13S-hydroxy-fatty acids
to two products: the 15,16-double bond of 13S-hydroxy
α-linolenic acid was oxidized stereospecifcally to the
15S,16R-cis-epoxide or the 13-hydroxyl was oxidized to
the 13-ketone. Products were identifed by UV, HPLC,
LC–MS, NMR and by comparison with authentic stand-
ards prepared for this study. The Pf01-cat displayed
similar activity. MAP-2744c oxidized 13S-hydroxy-lin-
oleic acid to the 13-ketone, and epoxidized the double
bonds to form the 9,10-epoxy-13-hydroxy, 11,12-epoxy-
13-hydroxy, and 9,10-epoxy-13-keto derivatives; equiva-
lent transformations occurred with 9S-hydroxy-linoleic
acid as substrate. In parallel incubations in the presence
of iodosylbenzene, human catalase displayed no activ-
ity towards 13S-hydroxy-linoleic acid, as expected from
the highly restricted access to its active site. The results
indicated that with suitable transformation to Compound
I, monooxygenase activity can be demonstrated by these
catalase-related hemoproteins with tyrosine as the proxi-
mal heme ligand.
Keywords Fusarium · Catalase · Lipoxygenase ·
Peroxidase · HODE · Iodosylbenzene
Abbreviations
PhIO
Iodosylbenzene
cAOS
COSY
Fg-cat
Catalase-related allene oxide synthase
Correlation spectroscopy
Fusarium graminearum catalase of the gene
FGSG_02217
H(P)ODE
H(P)OTE
HODE
Hydro(pero)xyoctadecadienoic acid
Hydro(pero)xyoctadecatrienoic acid
Hydroxyoctadecadienoic acid
Lipoxygenase
LOX
Electronic supplementary material The online version of this
article (doi:10.1007/s11745-017-4271-0) contains supplementary
material, which is available to authorized users.
MAP-2744c Mycobacterium avium ssp. paratuberculosis
catalase of gene MAP-2744c
mCPBA
Pf01-cat
Meta-chloroperoxybenzoic acid
Pseudomonas fuorescens Pf01 cata-
lase from the gene, accession number
WP_011333788.1
* Alan R. Brash
alan.brash@vanderbilt.edu
1
Department of Pharmacology and the Vanderbilt Institute
RP-HPLC
SP-HPLC
Reversed phase high pressure liquid
chromatography
Straight phase high pressure liquid
chromatography
of Chemical Biology, Vanderbilt, University School
of Medicine, Nashville, TN 37232, USA
2
Present Address: Department of Chemistry, Tallinn
University of Technology, Tallinn, Estonia
1 3

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Lipids (2017) 52:587–597
[14]; a P. fuorescens Pf01 catalase (37.5 kD, accession
number WP_011333788.1) with 32% sequence identity
to Fg-cat; and the M. avium ssp. paratuberculosis 33 kD
catalase (gene MAB-2744c) previously shown to exhibit
peroxidase activity using 13-hydroperoxy-linoleic acid
(13-HPODE) as oxidizing co-substrate.
Introduction
A growing number of catalase-related hemoproteins are
being identifed that catalyze tightly controlled transfor-
mations of fatty acid hydroperoxides; the resulting prod-
ucts include allene oxides, other epoxy derivatives, alde-
hydes formed by chain cleavage, or novel syntheses such
as formation of a bicyclobutane fatty acid (reviewed in
Ref. [1]). A fundamental difference between these fatty
acid peroxide-metabolizing catalases and the classic cata-
lase hemoproteins is the available access to their active
site. The classic catalases generally have highly restricted
access to the active site, which limits their reactivity to
the metabolism of very small substrates, usually hydro-
gen peroxide [2, 3]. By contrast, the structure of the fatty
acid peroxide-metabolizing catalase, while preserving the
central “catalase fold”, allows access of substrates with
C18 or C20 carbon chains [4, 5].
It has long been considered that the ability of the
cytochromes P450 to oxidize a range of small molecule
substrates is related to the special properties of the active
site environment with cysteine constituting the proximal
ligand to the heme [6, 7]. Certainly the thiolate ligand of
P450s helps confer potent oxidative capability in com-
parison to the commonly occurring hemoproteins with a
histidine proximal heme ligand [8]. Catalase hemopro-
teins have tyrosine as the proximal heme ligand, and as
indicated above, on account of the exceptionally limited
substrate repertoire of the classic catalases, it has not
been possible to examine the facility for catalyzing oxi-
dative transformations of “small molecules” (of molec-
ular weights ~100–500) within the catalase fold. The
availability of the relatively recently identifed fatty acid
peroxide-metabolizing catalases provides the opportunity
to conduct this type of experiment.
In earlier work the catalase-related allene oxide syn-
thase (cAOS) of the coral Plexaura homomalla was
shown to have P450-like oxidative capabilities when acti-
vated with the oxygen donor iodosylbenzene [9]. Iodo-
sylbenzene (PhIO) has long been employed in mechanis-
tic studies of cytochromes P450 as it is a single oxygen
donor that oxidizes the enzyme to Compound I and by-
passes the need for molecular oxygen and the reducing
equivalents from NADPH (e.g. Refs. [10–13]. When
activated using PhIO, the P. homomalla cAOS catalyzed
stereoselective epoxidations and hydroxylations of ara-
chidonic acid and 8R-hydroxy-eicosatetraenoic acid (the
hydroxy analog of its natural 8R-hydroperoxy substrate)
[9]. In the current work we further explored the oxida-
tive capacity of catalase-related hemoproteins using three
enzymes: Fg-cat, a 41 kD enzyme from F. graminearum
recently shown to act as a peroxidase using exclusively
the 13S-hydroperoxides of linoleic or α-linolenic acids
Materials and Methods
Materials
α-Linolenic, γ-linolenic and linoleic acids were purchased
from Nu-Chek Prep Inc. (Elysian, MN). Iodosylbenzene
was purchased from TCI America (Portland, OR, catalogue
no. I0072). The 13S-hydroxy derivatives were synthesized
from linoleic acid or α- or γ-linolenic acids using the soy-
bean LOX [15] and reduced subsequently with sodium
borohydride to the hydroxy derivatives, 13S-HODE, and
13S-HOTE-ω3 and 13S-HOTE-ω6. 9S-HODE was pre-
pared using potato 9S-LOX activity [16]. The 9- and
13-ketones were prepared by oxidation of the 9- and
13-hydroxy derivatives using activated manganese dioxide
or hematin treatment of the hydroperoxides.
Catalase‑Related Enzymes
Three of the four catalase enzymes used in this study were
cloned and expressed in E. coli as described previously: F.
graminearum gene FGSG_02217, encoding a 41.5 kD cata-
lase-related hemoprotein designated here as Fg-cat [14], M.
avium ssp. paratuberculosis gene MAP_2744c, encoding a
33 kD mini-catalase [5] and human catalase was similarly
expressed in E. coli [17]. The P. fuorescens Pf01 37.5 kD
catalase (accession number WP_011333788.1) was cloned
from the genomic DNA with an N-terminal His-tag (simi-
larly to the other three catalases used in this study) and
expressed in E. coli under the same conditions as used for
Fg-cat [14]. The four catalases were purifed by nickel affn-
ity chromatography and quantifed based on the absorbance
of the main Soret band (at ~405–410 nm) assuming a molar
extinction coeffcient of 100,000 M−1 cm−1
.
Incubations with Fg‑cat and 13S‑HOTE(ω3)
and 13S‑HOTE(ω6) in the Presence of Iodosylbenzene
Fresh 5 mM iodosylbenzene (PhIO) solution was prepared
as described previously [9]. Into 3 mL 50 mM Tris pH
7.5 + 150 mM NaCl buffer containing 50 μM iodosylben-
zene and 1 μM Fg-cat was added 50 μM 13S-HOTE(ω3)
or 13S-HOTE(ω6) substrate and incubated for 15 min at
room temperature. The remaining PhIO was then elimi-
nated by addition of 2–4 mg of solid sodium bisulfte and
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Lipids (2017) 52:587–597
589
the reactions were acidifed to pH 4.0 with 1 M KH2PO4
and 1 N HCl and extracted using a 1-cc HLB Oasis car-
tridge and eluted with MeOH. The eluted products were
taken to dryness and dissolved in a column solvent (metha-
nol/water/glacial acetic acid, 80:20:0.01, by volume) prior
to RP-HPLC analysis. Products were analyzed with this
solvent on RP-HPLC using a Waters Symmetry C18 5-µm
column (25 × 0.46 cm) with a fow rate at 1 mL/min. The
UV signals at 205, 220, 235, 280 nm were recorded on a
diode array (Agilent 1200 Series). About 0.5 mg of prod-
ucts formed from the 13S-HOTE incubations were isolated
on RP-HPLC for the 1H-NMR analysis. The other enzymes
studied, Pf01-cat, MAP2744c, and human catalase were
reacted with PhIO under similar conditions.
and a fow rate of 4 mL/min with on-line UV detection at
205 nm. The two 15,16-epoxide enantiomers, designated
AD1 and AD2 in order of elution (at 28 and 32 min reten-
tion times, respectively), were incompletely resolved due
to some tailing when amounts in the order of 1 mg were
injected. Subsequent analysis using an analytical Chiralpak
AD column (25 × 0.46 cm) with the same solvent system
and a fow rate of 1 mL/min showed AD1 (retention time
26.1 min) was pure while AD2 (at 29.8 min) contained
13% of AD1; this cross-contamination was removed by fur-
ther purifcation of AD2 on the semi-preparative Chiralpak
AD column.
The absolute confguration of the 15,16-epoxy enan-
tiomers was determined after chemical transformation to
16-HODE (methyl ester acetate derivative) using a protocol
reported for conversion of epoxyeicosatrienoic acids to the
corresponding hydroxy derivatives [19] and as described
before in detail for characterization of epoxy-linoleates
[20]. Each enantiomer of 16-HODE methyl ester (50 μg)
was dissolved in 50 µL acetonitrile and derivatized using
1 µL benzoyl chloride in the presence of 1 µL 1,8-diaz-
abicyclo[5.4.0]undec-7-ene (DBU) and a few grains of
4-dimethyl aminopyridine overnight at room temperature.
A blank reaction was prepared in parallel. The benzoyl
ester reactions were evaporated to dryness and applied to
a 0.5 g Bond-Elut silica cartridge in hexane followed by a
wash with 2 mL hexane and elution with 4 mL of 10% of
EtOAc in hexane. The benzoyl derivatives were then puri-
fed by RP-HPLC using a Waters Symmetry C18 5-µm col-
umn (25 × 0.46 cm), a solvent of MeOH/water/glacial ace-
tic acid (80:20:0.01 by volume) at a fow rate of 1 mL/min.
The collected benzoyl derivative of the 16-HODE methyl
ester from each 15,16-epoxy enantiomer was taken to dry-
ness, redissolved in acetonitrile and quantifed by UV–Vis
spectroscopy (ε ~25,000 at 235 nm) prior to the circular
dichroism spectroscopy (CD). The UV absorbance of the
benzoyl derivatives of 16-HODEs was adjusted to 0.8–
1.0 AU at 235 nm for optimal CD analysis and the spectra
recorded using a JASCO J-700 spectropolarimeter.
Isolation of the 15,16‑Epoxide of α‑Linolenic Acid
Racemic 15,16-epoxide from α-linolenic acid was iso-
lated from the mixture of epoxides formed by treatment of
α-linolenic acid with m-chloroperoxybenzoic acid as out-
lined as Scheme S1 in the Supplement. Briefy, α-linolenic
acid (25 mg) was epoxidized using a 1.5-fold molar excess
of m-chloroperoxybenzoic (mCPBA) in 2 mL dichlo-
romethane at room temperature for 30 min. The sample was
extracted with aqueous bicarbonate, washed with water and
the DCM phase taken to dryness under a stream of nitrogen
and stored in methanol. The mono-epoxides were frst iso-
lated as a group by RP-HPLC in several injections using a
Waters C18 Symmetry 5-μm column (25 × 0.46 cm) with
a solvent of MeOH/water/glacial acetic acid (85:15:0.01, by
volume) at a fow rate of 1 mL/min (retention time of epox-
ides, 8.5–10 min). The mixture of three mono-epoxides was
then resolved by SP-HPLC using an Alltech 5-μm silica
column (25 × 0.46 cm) with a solvent of hexane/isopro-
panol/glacial acetic acid (100:0.5:0.1, by volume) at a fow
rate of 1.5 mL/min; the epoxides are reported to elute in
the order 12,13, 15,16, and 9,10 [18], observed at retention
times of 23.3, 25.5, and 27.2 min, respectively. To confrm
the middle peak is the 15,16-epoxide, small aliquots of each
were tested for reaction with dilute soybean lipoxygenase
(UV assay scanning, for the appearance of a conjugated
diene chromophore [15]). This established that only the
middle peak with intact 9Z,12Z double bonds reacted read-
ily, in agreement with it being the 15,16-epoxide. Its struc-
ture was confrmed by 1H-NMR (Supplement Table S1).
Preparation of the Standards
of 13S‑Hydroxy‑15,16‑Epoxides
15S,16R- and 15R,16S-epoxy-octadeca-9Z,12Z-dienoic
acids were individually incubated with soybean 13S-lipox-
ygenase in the presence of reducing agent SnCl2 as
described previously [21]. In addition to hydro(pero)xy-
epoxy α-linolenic acid derivatives, monocyclic endoperox-
ide side-products formed and are reported in detail by Teder
et al. [21]. Enzymatically derived 15S,16R- and 15R,16S-
epoxy-13S-hydroxy derivatives were co-analyzed with the
product of Fg-cat from 13S-HOTE-ω3/PhIO on SP-HPLC
using a Beckman 5-µm silica column (25 × 0.46 cm) and a
Resolution and Absolute Confguration of the
Enantiomers of the 15,16‑Epoxide
The enantiomers of 15,16-cis-epoxy-octadeca-9Z,12Z-
dienoic acid were resolved using a semi-preparative Chi-
ralpak AD column (25 × 1 cm) with a solvent system of
hexane/MeOH/glacial acetic acid (100:2:0.05, by volume)
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Lipids (2017) 52:587–597
solvent of hexane/isopropanol (100:5, v/v) with a fow rate
at 1 mL/min (“Results”).
A
mAU
100
Fg-cat/PhIO + 13-HOTEω3
Unreacted
13S-HOTE (ω3)
80
60
40
20
0
HPLC of Epoxyalcohols from 9S‑HODE
and 13S‑HODE
O
Product 1
13-keto-18:3ω3
235 nm
280 nm
Epoxyalcohols and other products from the reactions
of 9S-HODE and 13S-HODE with MAP-2744c/PhIO
and with the epoxidizing reagent mCPBA were resolved
on SP-HPLC using a Thomson 5-μm silica column
(25 × 0.46 cm) with a solvent system of hexane/isopro-
panol/glacial acetic acid (100:2:0.01, by volume) for the
free acids and hexane/isopropanol 100:1 for the methyl
ester derivatives. The RP-HPLC analyses used a Waters
5-μm Symmetry C18 column (25 × 0.46 cm) with a
methanol-based solvent of MeOH/water/glacial acetic acid
(80:20:0.01, by volume), or an acetonitrile-based solvent of
CH3CN/water/glacial acetic acid (60:40:0.01, by volume)
run at a fow rate of 0.5 or 1 mL/min with on-line detection
at 205, 220, 235 and 270 nm.
B
160
Fg-cat/PhIO + 13-HOTEω6
Unreacted
13S-HOTE (ω6)
120
80
40
0
O
13-keto-18:3ω6
235 nm
280 nm
0
2
4
6
8
10
12
14
Retention time
Fig. 1 RP-HPLC analysis of products formed from a 13S-HOTE-ω3
and b 13S-HOTE-ω6 by Fg-cat in the presence of iodosylbenzene.
The reactions were analyzed using a Waters C18 Symmetry col-
umn (25 × 0.46 cm), a solvent of methanol/water/glacial acetic acid
(80:20:0.01 by volume) and a fow rate of 1 mL/min
LC–MS and NMR Analysis
Products formed from 13S-HOTE by Fg-cat in the pres-
ence of PhIO were separated using a Kinetex C18 2.6-µm
column (3 × 100 mm) with acetonitrile/water/glacial ace-
tic acid (45:55:0.01, by volume) column solvent at 0.4 mL/
min, and molecular weights were established from the
M–H anions measured by negative ion electrospray on a
LTQ1 ion trap LC–MS instrument.
just before the unreacted hydroxy substrate on RP-HPLC
and displayed the typical smooth absorbance chromophore
of a conjugated dienone (λmax 282 nm in the RP column
solvent for the product from the ω6 13-hydroxy substrate
and 284 nm for the product from ω3 13-hydroxy). Based
on the HPLC retention times, the UV spectra and in the
1H-NMR and 1H,1H COSY NMR spectra were obtained
on a Bruker 600 MHz spectrometer at 298 K. The parts/
million values are reported relative to residual nondeuter-
ated benzene (C6D6; δ = 7.15 ppm). All spectra were ana-
lyzed on Bruker TopSpin 3.0 software.
1
case of the product from 13S-HOTE-ω6, on the H-NMR
spectrum (Supplement, Table S1), the ~280 nm-absorbing
products eluting near the hydroxy starting materials were
identifed as the corresponding 13-ketone derivatives
(13-keto-octadeca-6Z,9Z,11E-trienoic acid and 13-keto-
octadeca-9Z,11E,15Z-trienoic acid, respectively).
Results
From 13S-HOTE-ω3, RP-HPLC analysis also indi-
cated the presence of an early-eluting peak, product 1,
with a retention time at 6.5 min, with 235 nm absorb-
ance and the UV spectrum typical of a conjugated diene.
No corresponding peak was observed from the incubation
with 13S-HOTE-ω6. The [M−1] value of product 1 was
determined by LC–MS as 309, corresponding to a molec-
ular weight of 310 and the addition of 16 amu compared
to the 13S-HOTE starting material (MW = 294). This fts
with the addition of oxygen across the 15,16-double bond
of 13S-HOTE with the retention of the 9Z,11E-conjugated
diene, and also would explain the lack of a related prod-
uct from the γ-linolenic acid derivative 13S-HOTE-ω6.
1H-NMR analysis proved the structure to be 13S-hydroxy-
15,16-cis-epoxy-octadeca-9Z,11E-dienoic acid (Supple-
ment Fig. S1 and Table S2). The COSY spectrum clearly
Reaction of Fg‑cat/PhIO with 13S‑HOTE‑ω3
and 13S‑HOTE‑ω6
As potential substrates for PhIO-activated Fg-cat, the
13S-hydroxy derivatives of α-linolenic and γ-linolenic
acids were selected for study because Fg-cat reacts spe-
cifcally with the 13S-hydroperoxides of unsaturated C18
fatty acids [14]. By itself Fg-cat is completely unreac-
tive with the hydroxy derivatives, as is their treatment
with PhIO alone (data not shown). On the other hand, the
two 13S-hydroxy substrates were converted to new prod-
ucts by Fg-cat in the presence of PhIO (Fig. 1). A single
product was formed from 13S-HOTE-ω6 and two from
13S-HOTE-ω3. The product they gave in common eluted
1 3

Lipids (2017) 52:587–597
591
established the covalent linkage of all functionalities
between H7 through C18. The chemical shifts and cou-
pling constants defned the confgurations of the 9,10-cis
and 11,12-trans double bonds and location of the hydroxyl
at C13. The COSY cross peaks and characteristic chemical
shifts confrmed the existence of a 15,16-epoxide, with the
coupling constant J15,16 = 4.2 Hz characteristic of the cis–
epoxy confguration (cf. trans-epoxy ~2.5 Hz [22]).
CD
∆ε
H
H
14
16
40
30
20
10
R
15
H
=
R
C2H5
H C2H5
220.5 nm
(+10.4)
16(S) AD2
16(R) AD1
H
0
-10
-20
H
14
239.5 nm
16
Stereochemical Analysis of Product 1
-30
-40
-50
15
H
=
C2H5
(-22.8)
R
R
C2H5 H
Product 1 gave a single peak on both RP-HPLC and SP-
HPLC, suggesting that it represents a single diastere-
omer. Given that the 13S-hydroxy structure is not directly
involved in the enzymatic reaction and can be assumed
to be unchanged, the question arises as to the confgura-
tion of the 15,16-epoxide? To address this issue, we pre-
pared the cis-mono-epoxides of α-linolenic acid, resolved
the mixture by RP-HPLC and SP-HPLC and identifed the
positional isomers (Supplement, Scheme S1) [18]. The 1H-
NMR spectra of these products are reported, although not
with assignments of specifc protons to the NMR signals
[18]; in the Supplement we include the full NMR spectrum
and COSY spectrum of the 15,16-epoxide with full assign-
ments (Supplement Fig. S2, Table S3). The enantiomers of
the 15,16-epoxide were then resolved using a Chiralpak
AD column (“Materials and Methods”) and designated as
AD1 and AD2 in order of elution.
AU
1.0
235 nm
UV
O
R =
O
0.8
0.6
0.4
0.2
0
200.0
220
240
260
280
300
Wavelength (nm)
Fig. 2 CD spectra of 16R- and 16S-HODE benzoyl derivatives
chemically derived from corresponding 15S,16R-(AD1) and 15R,16S-
epoxy-α-linolenic acid (AD2) as described in “Materials and Meth-
ods”
The AD1 and AD2 enantiomers of 15,16-epoxy-C18:2
were converted by chemical transformations [19] to the
corresponding 16-hydroxy-octadeca-9Z,12Z,14E-trienoates
(“Materials and Methods”). The 16-hydroxy enantiom-
ers were further converted to the 16-O-benzoate esters and
the circular dichroism (CD) spectra recorded [23–25]. This
allows assignment of the 16-HOTEs as the 16R-hydroxy
enantiomer from AD1 and 16S-hydroxy from AD2, Fig. 2.
Thus, the original cis-epoxides are assigned as 15S,16R-
epoxy (AD1) and 15R,16S-epoxy (AD2).
From this point, the AD1 and AD2 epoxides were
reacted with soybean LOX-1 and the resulting 13S-hydrop-
eroxides reduced to 13S-hydroxy. These 13S-hydroxy-
15,16-epoxides were then compared on SP-HPLC with
product 1 of Fg-cat, Fig. 3. (Note that the early-eluting
peaks in Fig. 3 are irrelevant to this analysis: they are
fatty acid endoperoxides resulting from the further trans-
formation of 13S-hydroperoxy-15,16-epoxides as char-
acterized previously [21]). The results in Fig. 3 show that
Fg-cat gives a single peak on SP-HPLC (Fig. 3a), the
two 13S-hydroxy-15,16-epoxide standards give separate
peaks at 7.4 min (15R,16S, AD2) and 7.65 min (15S,16R,
AD1) (Fig. 3b), and that, by co-injection, the Fg-cat prod-
uct co-chromatographs with the second of the two peaks
(Fig. 3c). Thus, the early-eluting Fg-cat Product 1 from
13S-HOTE-ω3 is identifed as 13S-hydroxy-15S,16R-
epoxy-octadeca-9Z,11E-dienoic acid, Scheme 1.
Reaction of Pf01‑cat/PhIO with 13S‑HOTEω3
and 13S‑HOTEω6
We selected the 41.5 kD P. fuorescens catalase (Pf01-
cat) for study because the histidine residue on the distal
heme is in the sequence context RATH, the Thr residue
adjacent to the distal His being characteristic of catalase
hemoproteins that can metabolize polyunsaturated fatty
acid hydroperoxides [1]. This P. fuorescens catalase
(Pf01-cat) has 32% sequence identity with Fg-cat and we
have unpublished work establishing that it exhibits per-
oxidase activity using fatty acid 13S-hydroperoxides as
the oxidizing co-substrate (similar to Fg-cat [14]). When
we tested the corresponding 13S-hydroxy analogues as
substrate for the Pf01-cat in the presence of PhIO, the
18:3ω3 analogue was completely transformed to the
15,16-epoxide with only a trace of 13-ketone formation
(Fig. 4a, Scheme 1). This incubation was conducted side-
by-side with the Fg-cat reaction shown in Fig. 1, indicat-
ing preferential transformation to the 15,16-epoxy deriva-
tive by the Pf01 catalase. On the other hand, metabolism
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592
Lipids (2017) 52:587–597
O
OH
13S
A
Product 1 of Fg-cat/PhIO+13S-HOTEω3
13S-HOTE
HO
200
mAU
150
Fusarium graminearum Fg-cat
Pseudomonas fluorescens Pfl01-cat
PhIO (FeV=O)
13S-hydroxy-
15,16-epoxide
(product 1)
O
O
OH
O
100
50
0
product 1
(main product)
HO
HO
13S
15S 16R
and
O
13-ketone
235 nm
13S-hydroxy-
15R,16S-epoxide
Scheme 1 Transformation of 13S-HOTE-ω3 by PhIO-activated Fg-
cat. The γ-linolenic acid analog, 13S-HOTE-ω6, lacks the 15,16-dou-
ble bond and is transformed only to the 13-ketone
B
160
Co-injection of 13S-OH-
15,16-epoxide standards
13S-hydroxy-
15S,16R-epoxide
120
A
80
40
mAU
100
Pfl01-cat/PhIO + 13-HOTEω3
80
60
40
20
Product 1
O
235 nm
0
13-keto-18:3ω3
13S-hydroxy-
15S,16R-epoxide
and
235 nm
280 nm
Co-injection of
standards + Product 1
C
0
B
13S-hydroxy-
15R,16S-epoxide
Product 1
400
300
200
Pfl01-cat/PhIO + 13-HOTEω6
Unreacted
13S-HOTE (ω6)
160
120
80
40
0
O
13-keto-18:3ω6
235 nm
280 nm
100
0
235 nm
14
0
2
4
6
8
10
12
2
4
6
8
10
Retention time
Retention time (min)
Fig. 4 RP-HPLC analysis of products formed from a 13S-HOTE-ω3
and b 13S-HOTE-ω6 by the P. fuorescens enzyme Pf01-cat in the
presence of iodosylbenzene. The HPLC conditions were identical to
those in Fig. 1
Fig. 3 Assignment of the absolute confguration of 13S-hydroxy-
15,16-epoxy-octadeca-9Z,11E-dienoic acid synthesized by Fg-cat/
PhIO. a the SP-HPLC chromatogram of 15,16-epoxy-13S-hydroxy-
octadeca-9Z,11E-dienoic acid by Fg-cat/PhIO; b the co-injection
of 13S-hydroxy derivatives formed from 15S,16R-epoxy-(AD1) or
15R,16S-epoxy-octadeca-9Z,11E-dienoic acid (AD2) by soybean
13S-lipoxygenase; c the co-injection of 15,16-epoxy-13S-hydroxy-
octadeca-9Z,11E-dienoic acid synthesized by Fg-cat/PhIO with both
diastereomers of 15,16-epoxy-13S-hydroxy-octadeca-9Z,11E-dienoic
acid. The products were analyzed as methyl esters and chromato-
graphed on a Beckman silica column (0.46 × 25 cm) using a hex-
ane/isopropanol (100:5; v/v) at a fow rate of 1 mL/min with the UV
detection at 235 nm. Early-eluting products on chromatograms b and
c are endoperoxide derivatives characterized by Teder et al. [21]
Reaction of MAP_2744c/PhIO and Human Catalase/
PhIO with HODE Substrates
The M. avium ssp. paratuberculosis 33 kD catalase was
shown previously to exhibit peroxidase activity using
13S-HPODE as oxidizing co-substrate [5]. When incu-
bated with the 13S-hydroxy derivative of linoleic acid
(13-HODE) the enzyme formed four products, in order
of elution from the RP-HPLC column assigned as the
9,10-epoxy-13-hydroxy derivative (product 1 in Fig. 5a),
predominantly (10:1) the 9R,10S,13S diastereomer as
established in comparison to authentic standards [16], the
9,10-cis-epoxy-13-ketone (product 2 in Fig. 5a), the minor
of the 13-hydroxy 18:3ω6 substrate was less extensive
than with Fg-cat (compare Fig. 4b with Fig. 1b). Struc-
tures of the 15,16-epoxy derivative and the 13-ketone
were confrmed by 1H-NMR.
1 3

Lipids (2017) 52:587–597
593
B
A
mAU
60
MAP-2744c/PhIO + 13-HODE
MAP-2744c/PhIO + 9-HODE
mAU
120
9-HODE
2
4
13-HODE
5
S
1
4
205 nm
235 nm
270 nm
205 nm
235 nm
270 nm
S
40
20
0
7
80
40
0
6
3
6
8
10
12
14
16
18
0
5
10
15
20
Retention time (min)
Retention time (min)
C
mAU
Human catalase/PhIO+ 13-HODE
13-HODE
300
S = solvent
injection artefact
205 nm
200
100
0
235 nm
270 nm
S
S
0
5
10
15
20
Retention time (min)
Fig. 5 RP-HPLC analysis of products formed from a 13S-HODE
(ω6) with MAP_2744c and b 9S-HODE with MAP_2744c, and c
13S-HODE with human catalase, each in the presence of iodosylb-
enzene. The HPLC conditions were the same as in Fig. 1. The four
products from 13S-HODE were identifed as (1) the 9,10-epoxy-
13-hydroxy derivative, (2) the 9,10-epoxy-13-keto derivative, (3)
the 11,12-epoxy-13-hydroxy derivative, and (4) the 13-ketone. From
9S-HODE the products are (5) the 9-hydroxy-12,13-epoxide, (6) the
9-keto-12,13-epoxide, (7) the 9-hydroxy-10,11-epoxide, and (8) the
13-ketone. The peaks marked “S” are UV artefacts related to injec-
tion of solvent
Scheme 2 Products formed
from 13S-HODE by the M.
avium ssp. paratuberculo-
sis 33 kD catalase MAP-
2744c. R=HO2C(CH2–)6 and
R’=C5H11
O
R'
R
(minor
3
1
4
OH
R'
pathway)
[Ox]
[O]
R
R
[O]
O
OH
13 R'
R'
R
R
9
O
O
OH
2
R'
O
[Ox]
13S-HODE
11,12-epoxy-13-hydroxy derivative (product 3 in Fig. 5a),
and the 13-ketone (product 4), Scheme 2. The equiva-
lent reactions occurred also using 9S-HODE as substrate
(Fig. 5b), forming the 9-hydroxy-12,13-cis-epoxide (prod-
uct 5), the 9-keto-12,13-cis-epoxide (product 6, identical
to the reported product of Fg-cat with 13S-HPODE [14]), a
minor 9-hydroxy-10,11-epoxy product 7, and the 9-ketone
(product 8). As a (hopefully!) negative control, a paral-
lel incubation was conducted with human catalase in the
presence of PhIO and 13S-HODE. Analysis by RP-HPLC
indicated no transformation of the 13-HODE substrate, as
expected for a conventional catalase enzyme with highly
restricted access to the heme active site (Fig. 5c).
The products from 9S-HODE and MAP-2744c/PhIO
were also separated using a different RP-HPLC sys-
tem (acetonitrile/water-based solvent as opposed to the
MeOH/water systems used in the other chromatograms)
and compared to the epoxyalcohols prepared by treat-
ment of 9S-HODE with the epoxidizing reagent mCPBA,
Fig. 6. The purely chemical transformation using mCPBA
gave four epoxyalcohols separated on SP-HPLC as two
9-hydroxy-10,11-epoxides (eluting at 10.2 and 15.5 min,
and designated as SP1 and SP2, respectively), followed
by two 9-hydroxy-12,13-epoxides (eluting at 21.4 and
23.0 min, and designated as SP3 and SP4, respectively,
Methods, and data not shown). As illustrated in Fig. 6a, on
1 3

594
Lipids (2017) 52:587–597
of which were not determined. In the enzyme reaction there
is no product corresponding to the mCPBA RP2, while
the minor product 7 corresponds to mCPBA RP3. Based
on published data on the relative polarity of 9S-hydroxy-
10,11-trans-epoxy-octadecenoates in which the more polar
isomer is of the 9,10-threo confguration and 9S,10R,11R
[26], the MAP-2744c epoxyalcohol 7 co-chromatographing
with mCPBA peak RP3 is the less polar diastereomer and
can be assigned as the 9,10-erythro isomer and 9S-hydroxy-
10S,11S-trans-epoxy in confguration. This stereochemical
assignment was confrmed by 1H-NMR of the SP1/RP3 and
SP2/RP2 products of the 9S-HODE/mCPBA reaction (Sup-
plement Fig. S3); the epoxide proton at the 10-carbon gives
a diagnostic J9,10 coupling constant of 3.1 Hz (erythro) in
the less polar peak SP1/RP3 and 4.6 Hz (threo) in SP2/
RP2, and in each case confrming the 10,11-trans-epoxide
(J10,11 = 2.3 Hz) [27, 28]. The results show that in regard
to the stereochemistry of epoxidation α,β to the 9-hydroxyl,
the MAP-2744c enzyme exhibits opposite stereoselectiv-
ity to a Saprolegnia parasitica hydroperoxide isomerase
that forms exclusively the 9,10-threo diastereomer [26,
28]). This difference in stereochemistry is true also for the
main epoxyalcohol product 1 formed from 13S-HODE,
9R,10S-epoxy-13S-hydroxy in our experiments com-
pared to 9S,10R-epoxy-13S-hydroxy when formed from
13S-HPODE by the S. parasitica isomerase [26]. Forma-
tion of the products from 9S-HODE and MAP-2744c/PhIO
are summarized in Scheme 3.
A
mAU
600
RP1
(SP3 and SP4) RP2
(SP2)
9S-HODE products using mCPBA
RP1, two 9-hydroxy-10E-12,13-epoxides
RP2 and RP3, 9-hydroxy,10,11-epoxy-(12Z)
RP3
400
200
(SP1)
unreacted
9S-HODE
(at 205 nm)
mCPBA
205 nm
0
B
mAU
MAP-2744c/PhIO+ 9-HODE
205 nm
235 nm
270 nm
5
300
6
8
unreacted
200
100
0
9S-HODE
7
0
10
20
30
40
60
50
Retention time (min)
Fig. 6 RP-HPLC analysis of 9S-HODE products formed by mCPBA
or MAP-2744c/PhIO. The chromatograms were run using a Waters
Symmetry 5-μm C18 column (25 × 0.46 cm) with acetonitrile/water/
glacial acetic acid (60:40:0.01, by volume) at 1 mL/min. a RP-HPLC
analysis of products formed by treatment of 9S-HODE with the
epoxidizing reagent mCPBA, producing three peaks of epoxy prod-
ucts on RP-HPLC (detected at 205 nm) and four peaks on SP-HPLC
(the latter not illustrated, see main text). b Oxidation of 9S-HODE by
MAP_2744c/PhIO. The products in order of elution were identifed
as (5) a 1:9 mixture of 9S-hydroxy-12,13-cis-epoxides correspond-
ing on SP-HPLC to the mCBPA products SP3 and SP4, respectively,
(7) 9S-hydroxy-10S,11S-trans-epoxy-octadeca-12Z-enoic acid, cor-
responding to mCPBA product RP3 (RP2 being absent in the enzy-
matic reaction), (6) 9-keto-12,13-cis-epoxy-octadeca-10E-enoic acid,
and (8) 9-keto-octadeca-10E,12Z-dienoic acid. A Waters Symmetry
5-μm C18 column (25 × 0.46 cm) was eluted with acetonitrile/water/
glacial acetic acid (60:40:0.01, by volume) at 1 mL/min
Discussion
When activated with PhIO, the 41 kD Fg-cat and the 37.5
kD Pf01-cat catalyzed a positional- and stereo-specifc
epoxidation of the 15,16 double bond of α-linoleic acid-
derived 13S-HOTE. The stereospecifc epoxidation of
13S-HOTE by Fg-cat matches the catalytic activity of the
PhIO-activated P. homomalla cAOS with 8R-HETE [9]. In
the current work we also found that the 33-kD mini-cata-
lase of M. avium ssp. paratuberculosis [5] will epoxidize
the cis double bond of 9S- and 13S-HODE. Regarding ole-
fn epoxidation, there is some debate in the P450 litera-
ture as to the involvement of Compound I or Compound 0
RP-HPLC the mCPBA products gave only three peaks, the
frst containing a 1:1 mixture of SP3 and SP4, the late-elut-
ing 9-hydroxy-12,13-epoxides on SP-HPLC.
From the MAP-2744c/PhIO reaction with 9S-HODE
the prominent product 5 (Fig. 6b) resolved on SP-HPLC
analysis as a 1:9 mixture of the SP3 and SP4 diastereomers,
respectively (data not shown), the absolute confgurations
Scheme 3 Products formed
from 9S-HODE by the M.
avium ssp. paratuberculo-
sis 33 kD catalase MAP-
2744c. R=HO2C(CH2–)6 and
R'
R
(minor
O
HO
pathway)
7
R'
R'
[Ox]
[O]
R
R
[O]
HO
O
9
R'
13
R’=C5H11
R'
5
8
R
R
O
O
HO
6
9S-HODE
[Ox]
O
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Lipids (2017) 52:587–597
595
Scheme 4 Mechanisms of
PhIO-activated Fg-cat trans-
formation of 13S-HOTE-ω3.
(13S-HOTE-ω6 is transformed
through the ketone pathway
only)
2nd H-abstraction
FeIII + H2O
Ketone pathway
HO
FeIV-OH
1st H-abstraction
FeV=O
FeIV-OH
O
13-ketone
HO
H
15 16
FeIII
13
Fe
O
HO
H
O
HO
H
13
FeV=O
15S,16R-
epoxide
Epoxide pathway
(the Fe-peroxy precursor of Cmpd I in the P450 catalytic
cycle) as the active enzyme species [29]. In our case the
use of the single oxygen donor PhIO is compatible only
with oxidation via Compound I. Compared to oxidation
of alkanes, olefn epoxidation is a relatively “easy” trans-
formation and is common not only among P450s [29], but
also for the heme-thiolate unspecifc fungal peroxygenases
(EC 1.11.2.1) [30], and the plant heme peroxygenases [31].
Dioxygenation and monooxygenation activity in the pres-
ence of hydrogen peroxide or cumene peroxide is also
exhibited by hemoproteins such as myoglobin with an
accessible active site, forming H(P)ODE or epoxides from
linoleic acid [32, 33]. Notably, in our experiments no reac-
tion whatsoever occurred using human catalase/PhIO/13-
HODE in accord with the narrow channel limiting access to
the distal heme of conventional catalases [3].
A second product from 13S-HOTEs and 9- and
13-HODEs, also of mechanistic interest, is oxidation of
the hydroxyl to the corresponding ketone. (By contrast,
the PhIO-activated P. homomalla cAOS with 8R-HETE
did not form any ketones [9]). The enzymatic oxidation
of substrate hydroxyl to ketone and not involving dehy-
drogenases is the topic of several mechanistic studies (e.g.
[34–36]. Two main routes to the ketone are usually con-
sidered, one involving mono-oxygenation of the alcohol to
the gem-diol followed by loss of H2O, and alternatively a
double hydrogen abstraction mechanism, from the OH and
the geminal H, leaving the ketone as product. Some of the
lines of evidence in favor of the gem-diol mechanism use
initial 18O-labeling of the alcohol and interpreting a loss of
18O in the product as evidence of the 50:50 loss of oxygen
expected from the gem-diol intermediate [34]; nonethe-
less, interpretation of such studies may be compromised by
facile oxygen exchange from the resulting ketone. For the
ketone formation by Fg-cat, an attractive feature of the dual
H-abstraction mechanism is its parallel to the mechanism
we proposed in the transformation of 13S-hydroperoxides
[14]; Fg-cat will rearrange 13-hydroperoxy-linoleic acid
to the 9-oxo-12,13-epoxide derivative and explaining how
the 9-oxo moiety arises most likely involves transformation
of a 9-hydroperoxide intermediate to the 9-ketone via dual
H-abstraction [14].
Also in favor of the dual hydrogen abstraction one can
recall the uncommon, although well documented alkane
desaturase activities of certain cytochrome P450s [37–39].
The P450-catalyzed desaturation of alkanes entails ini-
tial H-abstraction by Compound I as in the conventional
hydroxylation pathway. However, instead of being fol-
lowed by oxygen rebound and thus hydroxylation of the
substrate, a second H-abstraction is achieved by the Com-
pound II intermediate, producing the alkene as product, the
latter step having mechanistic parallels to the mechanism of
fatty acid AOS enzymes [40]. Thus, we propose the mecha-
nisms of epoxidation and ketone formation as illustrated in
Scheme 4. For the sake of simplicity not all potential inter-
mediates in these transformations are shown. For example,
in the ketone pathway the order of hydrogen abstractions
could be reversed, and in the epoxide pathway there are
several ways to represent the interaction of the ferryl oxy-
gen with the double bond [29]. The pathways illustrated
suggest a similar steric relationship of the enzyme (Fe=O)
to the reacting substrate, removing the geminal H13 in the
ketone pathway and bonding with the reacting substrate on
the same side of the fatty acid molecule (Scheme 3).
As noted in the introduction, the possibilities for catalase-
related hemoproteins to catalyze fatty acid oxygenations
are restricted to the enzymes such as Fg-cat that can allow
access of medium sized substrates to the active site. We show
here that under suitable circumstances alkene epoxidation is
a possibility. In our experiments the heme iron was activated
artifcially using PhIO and whether there is a physiologi-
cal oxidant that could perform a similar function is perhaps
doubtful. Fg-cat shows minimal reaction with hydrogen per-
oxide and the fatty acid 13S-hydroperoxides that are sub-
strates and potential oxidants induce rapid turnover with for-
mation of new fatty acid derivatives and enzyme inactivation
[14]. Nonetheless, our fndings help consolidate the view
that the heme center of catalases with its tyrosine proximal
heme ligand has the potential to catalyze mono-oxygenation
of alkenes, and it some cases fatty acid hydroxylation too [9].
1 3

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Lipids (2017) 52:587–597
derivatives of 9-hydroperoxy-linoleic acid from hematin and
enzymatic synthesis. Chem Phys Lipids 167–168:21–32
17. Gao B, Boeglin WE, Brash AR (2008) Role of the conserved
distal heme asparagine of coral allene oxide synthase (Asn137)
and human catalase (Asn148): mutations affect the rate but not
the essential chemistry of the enzymatic transformations. Arch
Biochem Biophys 477:285–290
Acknowledgements This work was supported in part by National
Institutes of Health Grants GM-074888 and GM-15431 (to A. R. B.)
and by the Institutional Research Funding IUT19-9 of the Estonian
Ministry of Education and Research and the Estonian Science Foun-
dation Grant 9410 (to Professor Nigulas Samel).
Compliance with ethical standards
18. Cui PH, Duke RK, Duke CC (2008) Monoepoxy octadecadi-
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characterization. Chem Phys Lipids 152:122–130
Confict of interest The authors declare no conficts of interest.
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