Molecular characterization of NbEH1 and NbEH2, two epoxide hydrolases from Nicotiana benthamiana

Article abstract of DOI:10.1016/j.phytochem.2013.02.020

Plant epoxide hydrolases (EH) form two major clades, named EH1 and EH2. To gain a better understanding of the biochemical roles of the two classes, NbEH1.1 and NbEH2.1 were isolated from Nicotiana benthamiana and StEH from potato and heterologously expressed in Escherichia coli. The purified recombinant proteins were assayed with a variety of substrates. NbEH1.1 only accepted some aromatic epoxides, and displayed the highest enzyme activity towards phenyl glycidyl ether. In contrast, NbEH2.1 displayed a broad substrate range and similar substrate specificity as StEH. The latter enzymes showed activity towards all fatty acid epoxides examined. The activity (V_max) of NbEH1.1 towards phenyl glycidyl ether was 10 times higher than that of NbEH2.1. On the contrary, NbEH2.1 converted cis-9,10-epoxystearic acid with V_max of 3.83 μmol min mg^-1 but NbEH1.1 could not hydrolyze cis-9,10- epoxystearic acid. Expression analysis revealed that NbEH1.1 is induced by infection with tobacco mosaic virus (TMV) and wounding, whereas NbEH2.1 is present at a relatively constant level, not influenced by treatment with TMV and wounding. NbEH1.1 transcripts were present predominantly in roots, whereas NbEH2.1 mRNAs were detected primarily in leaves and stems. Overall, these two types of tobacco EH enzymes are distinguished not only by their gene expression, but also by different substrate specificities. EH1 seems not to participate in cutin biosynthesis and it may play a role in generating signals for activation of certain defence and stress responses in tobacco. However, members of the EH2 group hydrate fatty acid epoxides and may be involved in cutin monomer production in plants.

Full text of DOI:10.1016/j.phytochem.2013.02.020

Phytochemistry 90 (2013) 6–15
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Molecular characterization of NbEH1 and NbEH2, two epoxide
hydrolases from Nicotiana benthamiana
⇑
Fong-Chin Huang, Wilfried Schwab
Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, Freising D-85354, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Plant epoxide hydrolases (EH) form two major clades, named EH1 and EH2. To gain a better understand-
ing of the biochemical roles of the two classes, NbEH1.1 and NbEH2.1 were isolated from Nicotiana benth-
amiana and StEH from potato and heterologously expressed in Escherichia coli. The purified recombinant
proteins were assayed with a variety of substrates. NbEH1.1 only accepted some aromatic epoxides, and
displayed the highest enzyme activity towards phenyl glycidyl ether. In contrast, NbEH2.1 displayed a
broad substrate range and similar substrate specificity as StEH. The latter enzymes showed activity
towards all fatty acid epoxides examined. The activity (Vmax) of NbEH1.1 towards phenyl glycidyl ether
was 10 times higher than that of NbEH2.1. On the contrary, NbEH2.1 converted cis-9,10-epoxystearic acid
Received 29 January 2013
Accepted 28 February 2013
Available online 3 April 2013
Keywords:
Epoxide hydrolase
Peroxygenase
Nicotiana benthamiana
Epoxide
with Vmax of 3.83 l
mol min mgꢀ1 but NbEH1.1 could not hydrolyze cis-9,10-epoxystearic acid. Expression
analysis revealed that NbEH1.1 is induced by infection with tobacco mosaic virus (TMV) and wounding,
whereas NbEH2.1 is present at a relatively constant level, not influenced by treatment with TMV and
wounding. NbEH1.1 transcripts were present predominantly in roots, whereas NbEH2.1 mRNAs were
detected primarily in leaves and stems. Overall, these two types of tobacco EH enzymes are distinguished
not only by their gene expression, but also by different substrate specificities. EH1 seems not to partic-
ipate in cutin biosynthesis and it may play a role in generating signals for activation of certain defence
and stress responses in tobacco. However, members of the EH2 group hydrate fatty acid epoxides and
may be involved in cutin monomer production in plants.
Diol
Triol
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
NtEH1, a cytosolic EH from Nicotiana tabacum, was induced during
the hypersensitive response to tobacco mosaic virus but was not
Epoxide hydrolases (EH, EC 3.3.2.3) catalyze the conversion of
epoxides to their corresponding vicinal diols. These enzymes are
found in various species of mammals, insects, fungi, bacteria and
plants. The functions of mammalian EHs are diverse, including reg-
ulation of inflammation, xenobiotic detoxification and drug metab-
olism (Newman et al., 2005; Morisseau and Hammock, 2005). For
microorganisms, EHs seem important in the catabolism of specific
carbon sources from natural sources, such as limonene (Van der
Werf et al., 1999). Plant EHs appear to play a role in the biosynthe-
sis of monomers of cutin, a polymer that accumulates in cell walls
of damaged tissues (Blée and Schuber, 1993; Blée, 1998). Cutin is
important in plant development, protection from environmental
stresses, and disease resistance (Kato et al., 1983; Kiyosue et al.,
1994; Pinot et al., 2000). In addition to its participation in cutin
biosynthesis, EH may have other roles during plant defence re-
sponses. Expression of a cytosolic EH of Citrus jambhiri increased
upon inoculation with the pathogen Alternaria alternate, but tran-
scripts were not detected in healthy leaves (Gomi et al., 2003).
affected in the compatible interaction (Guo et al., 1998). Resistant
plants often exhibit a rapid and localized cell death at the site of
pathogen infection, termed the hypersensitive response. This rapid
cell death process is thought to help prevent pathogen multiplica-
tion and spread. NbEH1.1 in Nicotiana benthaminana showed in-
creased expression during compatible interactions with the
hemibiotrophic fungal pathogens, Colletotrichum destructivum and
Colletotrichum orbiculare, and the hemibiotrophic bacterial patho-
gen, Pseudomonas syringae pv. tabaci (Wijekoon et al., 2008). In
addition, epoxides are usually reactive causing toxicity to cells.
Hence, another role for EHs in plants would be to break down
epoxides accumulating during stress into less reactive compounds
(Murray et al., 1993).
In plants, EHs have been characterized from several organisms,
such as broad bean (Hamberg and Fahlstadius, 1992), soybean
(Blée and Schuber, 1992a,b; Blée and Schuber, 1995; Arahira
et al., 2000; Blée et al., 2005), Arabidopsis (Kiyosue et al., 1994), po-
tato (Stapleton et al., 1994; Morisseau et al., 2000; Elfström and
Widersten, 2005; Mowbray et al., 2006), tobacco (Guo et al.,
1998), Brassica napus (Bellevik et al., 2002), and Euphorbia lagascae
(Edqvist and Farbos, 2003). The epoxide hydrolase from potato
⇑
Corresponding author. Tel.: +49 8161 712912; fax: +49 8161 712950.
E-mail address: schwab@wzw.tum.de (W. Schwab).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.02.020

F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
7
(StEH1) is the best characterized plant EH. The expression of StEH1
was found to be regulated by both developmental and environ-
mental signals. Its mRNA was observed to accumulate on wound-
ing and application of exogenous methyl jasmonate (Stapleton
et al., 1994). The polypeptide encoded by StEH1 is similar to mam-
malian soluble EH both in sequence and enzymatic properties. The
recombinant StEH1 enzyme hydrolyzed a commonly used diagnos-
tic substrate for the soluble form of mammalian EH. Inhibitor pro-
files of the recombinant StEH1 and mammalian soluble EH were
also similar (Stapleton et al., 1994; Morisseau et al., 2000). Another
well characterized plant EH is soybean fatty acid EH (Blée and
Schuber, 1992a,b, Blée and Schuber, 1995; Blée et al., 2005). It is
quite specific for fatty epoxides. This enzyme hydrates preferen-
tially 9(R),10(S)-epoxy-12(Z)-octadecenoic and 12(R),13(S)-epoxy-
9(Z)-octadecenoic acid (Blée and Schuber, 1992a).
Comparison of the protein sequences of EHs from plants re-
vealed two major clades, which are named EH1 and EH2 (Wijekoon
et al., 2008). Both EH1 and EH2 type sequences have been found in
many plants, such as Allium cepa, Hordeum vulgare, Nicotiana benth-
amiana, Nicotiana tabacum, Capsicum annuum, Solanum tuberosum,
Oryza sativa and Triticum aestivum. However, an exception was
the Arabidopsis thaliana genome, which contains 33 EH genes that
are all of the EH2 type (Wijekoon et al., 2008). Four EHs have been
found in N. benthamiana, namely NbEH1.1 and NbEH1.2 of the EH1
clade and NbEH2.1 and NbEH2.2 of the EH2 clade (Wijekoon et al.,
2008, 2011). Motif analysis among 30 EH1 and EH2 from Solana-
ceaeous plants showed differences primarily in the lid region
around the catalytic site. In silico models of 3D structures also
showed significant differences in the lid region between NbEH1.1
and NbEH2.1 (Wijekoon et al., 2011). NbEH2.1 and NbEH2.2 have
a predicted peroxisomal targeting sequence, catalytic triad, and
structural similarities to a potato epoxide hydrolase (StEH1)
(Mowbray et al., 2006; Thomaeus et al., 2008). Besides, the gene
expression levels of NbEH1.1, NbEH1.2, NbEH2.1, and NbEH2.2 in re-
sponse to different pathogens have been analyzed (Wijekoon et al.,
2008, 2011). The results showed that the expression pattern of
NbEH1 was different from that of NbEH2, demonstrating special-
ization among EH genes in basal resistance.
lyzed in detail. In addition, the expression patterns of these two EH
isoforms were examined to disclose their potential biological
functions.
2. Results and discussion
2.1. Heterologous expression and purification of NbEH1.1, NbEH2.1
and StEH
Full-length cDNAs of NbEH1.1 and NbEH2.1 genes were isolated
from leaf of N. benthamiana, whereas that of StEH gene was isolated
from potato tuber. The open reading frames of NbEH1.1, NbEH2.1
and StEH were cloned into the pQE30 vector for expression in
E. coli to characterize the catalytic activities of the encoded pro-
teins. The protein sequence length of NbEH1.1, NbEH2.1 and StEH
is 311, 315 and 321 amino acids, respectively. NbEH1.1 is 35% and
34% identical to NbEH2.1 and StEH, respectively, while NbEH2.1 is
61% identical to StEH. Furthermore, the recombinant proteins were
purified using His-tag purification system. The proteins were sep-
arated on a 10% SDS–polyacrylamide gel and visualized by Coo-
massie staining. The stained gel showed the recombinant
NbEH1.1, NbEH2.1, and StEH proteins with molecular size of ca.
37.6 kDa, 37.7 kDa, and 38.4 kDa, respectively (Fig. 1A–C). A large
amount of NbEH1.1 protein was expressed and purified to homo-
geneity. Although NbEH2.1 protein was less expressed, it could
be purified to homogeneity. However, many other proteins were
still detected in the purified fractions of StEH. An another vector
pET29a(+) was also used to express NbEH2.1 and StEH proteins,
however, less amount and lower purity of proteins were obtained
(data not shown).
2.2. Functional analysis of NbEH1.1, NbEH2.1 and StEH proteins
Purified EH proteins were used for enzyme activity assays to-
wards a variety of substrates. Product formation was confirmed
by LC–MS and GC–MS. The used substrates can be grouped into
four classes: aromatic epoxides, terminal aliphatic epoxides, limo-
nene oxides and fatty acid epoxides (Supplementary Fig. S1A–D).
All substrates were also incubated with negative (empty vector)
controls. No diol products were formed in the control experiments.
NbEH1.1 only showed activity with artificial substrates such as
phenyl glycidyl ether and styrene oxide (Table 1). Phenyl glycidyl
ether was the preferred substrate and the activity of NbEH1.1 to-
wards this aromatic epoxide was even higher than that of NbEH2.1.
To gain a better understanding of the molecular mechanism and
the biochemical roles of EH1 and EH2 in plants, and to explore po-
tential biotechnological applications, NbEH1.1 and NbEH2.1 were
isolated from N. benthamiana as well as StEH from potato and het-
erologously expressed in Escherichia coli. The purified recombinant
proteins were assayed for their enzymatic activities with a variety
of epoxide substrates and the formation of fatty acid diols was ana-
A
B
C
M
Cr FT F3 F4 F5 F6 F7
M
Cr FT F3 F4 F5 F6
M
Cr FT F3 F4 F5 F6
kDa
250
130
95
72
55
36
28
Fig. 1. Coomassie-stained polyacrylamide gel analysis of recombinant EH proteins expressed in the pQE30 vector system. (A) Purification of NbEH1.1 protein: purified protein
fractions (F3–F6). (B) Purification of NbEH2.1 protein: purified protein fractions (F3–F7). (C) Purification of StEH protein: purified protein fractions (F3–F6). M: Marker; Cr:
total protein; FT: flow through. The purified proteins were indicated by arrows (?).

8
F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
Table 1
The catalytic activities of recombinant NbEH1.1, NbEH2.1, and StEH towards various epoxide substrates. Activities were determined by LC–MS and GC–MS. Values are the means
of two or three separate determinations. Measurements were performed with purified protein. Epoxide structures are shown in Supplementary Fig. S1. The lower detection limit
of reaction progress is 0.001
l .
mol minꢀ1 mgꢀ1
Substrates
NbEH1 (
l
mol minꢀ1 mgꢀ1
)
NbEH2 (
l
mol minꢀ1 mgꢀ1
)
StEH (l )
mol minꢀ1 mgꢀ1
cis-Stilbene oxide
trans-Stilbene oxide
trans-1,3-Diphenyl-2,3-epoxypropan-1-one
Phenyl glycidyl ether
–
–
–
–
0.003 0.0002
0.244 0.016
0.009 0.0002
0.235 0.003
0.068 0.004
0.043 0.002
0.022 0.001
0.268 0.019
Styrene oxide
0.114 0.005
0.030 0.0002
0.749 0.049
1,2-Epoxypentane
Glycidyl isopropyl ether
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
cis-
cis-
D
-Limonene-1,2-oxide
-Limonene-1,2-oxide
D
trans-
trans-
D
-Limonene-1,2-oxide
L-Limonene-1,2-oxide
cis-9,10-Epoxystearic acid
1.142 0.017
0.552 0.217
0.699 0.275
0.026 0.003
0.532 0.072
1.145 0.092
1.305 0.216
1.504 0.249
0.244 0.048
0.560 0.108
cis-9,10-Epoxy-12-octadecenoic acid
cis-12,13-Epoxy-9-octadecenoic acid
12,13-Epoxy-9-HOD
cis-11,12-Epoxyeicosanoic acid
NbEH1.1 protein preferred alkaline (pH 10) and NbEH2.1 neutral
pH values (pH 7–8). Both enzymes preferred relative high temper-
ature. A EH enzyme cloned from Brassica napus (Bellevik et al.,
2002) whose amino acid sequence is 57% identical to NbEH2.1 also
preferred high temperature (55 °C) and preferred neutral pH values
(pH 6-7) like NbEH2.1. The activity (Vmax) of NbEH1.1 towards phe-
nyl glycidyl ether was 10 times higher than that of NbEH2.1. On
the contrary, NbEH2.1 converted cis-9,10-epoxystearic acid effi-
ciently but NbEH1.1 could not hydrolyze cis-9,10-epoxystearic
acid.
Table 2
Comparison of kinetic properties for recombinant NbEH1.1 and NbEH2.1 measured
using phenyl glycidyl ether and rac cis-9,10-epoxystearic acid as substrates. Values
are the means of three separate determinations. One unit of activity (U) corresponds
to the amount of enzyme that converts 1 lmol of substrate per minute.
NbEH1.1
NbEH2.1
Substrate phenyl glycidyl ether
Temperature optimum
pH optimum
50 °C
10
2.30 0.28
1.30 0.04
0.58 0.06
40 °C
7
0.51 0.05
0.13 0.01
0.26 0.02
Km (mM)
Vmax (U mgꢀ1
)
Vmax/Km (U mgꢀ1 mMꢀ1
)
2.3. Metabolism of fatty acids by PXG and EH
Substrate rac cis-9,10-epoxystearic acid
Temperature optimum
pH optimum
No activity
–
–
–
–
–
40 °C
pH 8
0.17 0.02
3.83 0.40
23.38 2.29
The peroxygenase (PXG) cascade constitutes an additional
branch to the lipoxygenase pathway (Blée and Schuber, 1993;
Blée, 1998). PXG catalyzes, in higher plants, the efficient hydroper-
oxide-dependent epoxidation of unsaturated fatty acids such as
oleic and linoleic acid (Blée and Schuber, 1990; Hamberg and Ham-
berg, 1990; Hamberg and Fahlstadius, 1992; Blée, 1998). In addi-
tion to PXG, this cascade also involves EH that catalyzes the
trans-hydration of fatty acid cis-epoxides into their corresponding
dihydrodiols. To gain more knowledge about the metabolism path-
way of fatty acids catalyzed by PXG and EH, various fatty acids and
fatty acid hydroperoxide were added to reaction mixtures that
contained PXG and EH. A full-length PXG cDNA, named SlPXG
was recently cloned from tomato and expressed in yeast (Agho-
fack-Nguemezi et al., 2011).
At first, fatty acids were incubated with SlPXG yeast extract at
30 °C in the presence of hydrogen peroxide for 30 min, then puri-
fied EH was added and incubated for an additional 30 min. Hydro-
gen peroxide served as an effective oxygen donor in the
peroxygenase reaction. However, for fatty acid hydroperoxide sub-
strates no additional hydroperoxide was required to be added to
the reaction. Oleic acid, cis-11-eicosenoic acid, linoleic acid, and
9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9(S)-HPOD)
were used as substrates. SlPXG converted oleic acid and cis-11-
eicosenoic acid into cis-9,10-epoxystearic acid and cis-11,12-epox-
yeicosanoic acid, respectively. The formation of epoxides by SlPXG
from linoleic acid, and 9(S)-HPOD is described below. Moreover, all
fatty acid epoxides formed by SlPXG could be further converted
into corresponding diols by NbEH2.1 and StEH. However, no fatty
acid epoxides examined could be hydrolyzed into diols by
NbEH1.1. The yeast cells containing empty vector (negative con-
trol) were also incubated with all fatty acid epoxides examined.
No corresponding diol products were detected in the control
experiments.
Km (mM)
Vmax (U mgꢀ1
)
Vmax/Km (U mgꢀ1 mMꢀ1
)
Fatty acid epoxides were not hydrolyzed by NbEH1.1 and no diol
products were detected even after 20 h incubation. In contrast,
the EH2 clade members NbEH2.1 and StEH showed similar enzyme
activities towards all substrates examined and a broad substrate
range. In contrast to NbEH1.1, NbEH2.1 and StEH showed high en-
zyme activity towards fatty acid epoxides. 1,2-Epoxypentane, glyc-
idyl isopropyl ether, and any limonene epoxides were not
converted by the three enzymes. NbEH1.1 and NbEH2.1 showed
clear differential substrate specificity. This may indicate that they
play diverse biochemical roles in plants.
In order to gain more details about the catalytic properties of
NbEH1.1 and NbEH2.1, the kinetic characteristics of phenyl glyc-
idyl ether and cis-9,10-epoxystearic acid were determined for
these two enzymes (Table 2). The enzymatic activities of NbEH1.1
and NbEH2.1 were analyzed at different pH values (pH 2–12) and
temperatures (15–60 °C) to find out the optimal catalytic condi-
tions (Supplementary Fig. S2). The recombinant NbEH1.1 enzyme
has a temperature and pH optimum at 50 °C and pH 10, respec-
tively with phenyl glycidyl ether as substrate. The Km for this sub-
strate was 2.3 mM (Table 2). For the same substrate NbEH2.1 has a
temperature optimum at 40 °C and a pH optimum of pH 7. The Km
for this substrate was 0.51 mM. In addition, the recombinant
NbEH2.1 enzyme has a temperature optimum at 40 °C and a pH
optimum of pH 8 with cis-9,10-epoxystearic acid as a substrate.
The Km for this substrate was 0.17 mM (Supplementary Fig. S3).
However, no product was formed from cis-9,10-epoxystearic acid
by NbEH1.1, even after 20 h incubation. Taken together, the

F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
9
A
B
E
C
2.5
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
II
I
III
IV
8
6
4
2
m/z 313 [M-H]-
m/z 295 [M-H]-
1.5
m/z 183
m/z 195
(MS2 313 183)
1.0
(MS2 295 195)
0.5
0.0
1.5 m/z 201
m/z 171
(MS2 313
201)
(MS2 295 171)
1.0
0.5
0.0
34
35
36
37
38
39
39 Time (min) 42
36
37
38
40
41
43
44
45
Time (min)
D
8
6
4
2
8
6
4
2
III
IV
m/z 313 [M-H]-
m/z 313 [M-H]-
2.5
2.0
1.5
1.0
0.5
0.0
2.5
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
m/z 183
m/z 183
(MS2 313 183)
(MS2 313 183)
0.0
1.5
m/z 201
m/z 201
(MS2 313 201)
(MS2 313 201)
1.0
0.5
0.0
34
35
36
37
38
39
34
35
36
37
38
39
Time (min)
Time (min)
COOH
Linoleic acid
PXG
45 %
PXG
55 %
O
COOH
-H
195
O
12,13-Epoxy-9(Z)-octadecenoic acid (I)
COOH
-H
EH
171
9,10-Epoxy-12(Z)-octadecenoic acid (II)
OH
HO
COOH
-H+H
12,13-Dihydroxy-9(Z)-octadecenoic acid (III)
183
EH
OH
HO
COOH
-2H
201
9,10-Dihydroxy-12(Z)-octadecenoic acid (IV)
Fig. 2. LC–MS analysis of products formed by incubation of linoleic acid with (A) SlPXG, (B) SlPXG and NbEH1.1, (C) SlPXG and NbEH2.1, and (D) SlPXG and StEH. (E)
Metabolism of linoleic acid by PXG and EH. The PXG products 12,13-epoxy-9(Z)-octadecenoate (I) and 9,10-epoxy-12(Z)-octadecenoate (II) were monitored at m/z of 195 and
171, respectively (A). For detection of EH products 12,13-dihydroxy-9(Z)-octadecenic acid (III) and 9,10-dihydroxy-12(Z)-octadecenic acid (IV), m/z of 183 and 201 were
looked at, respectively (B, C, D). PXG: peroxygenase; EH: epoxide hydrolase.

10
F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
A
10
5
TriHome
10a
0
PXG+9-HPOD (acid-
hydrolysis)
7a 11a
10
V
V
6a 10a
5
0
PXG+9-HPOD
10
5
0
PXG+NbEH1+9-HPOD
PXG+NbEH2+9-HPOD
PXG+StEH+9-HPOD
10
V
V
5
0
10
VII
VI
5
0
10
5
0
VI VII
33.5
34.0
34.5
35.0
Time (min)
B
OOH
R
9(S)-HPOD
PXG
O
OH
R
12(R),13(S)-epoxy-9(S)-HOD (V)
EH
EH
OH
HO
OH
R
OH
HO
OH
R
9(S),12(R),13(R)-trihydroxy-10(E)-
octadecenoic acid (VI)
9(S),12(S),13(S)-trihydroxy-10(E)-
octadecenoic acid (VII)
Fig. 3. GC–MS analysis of products generated by incubation of 9(S)-HPOD with SlPXG and various EHs. (A) Products were isolated by extraction with ethyl acetate,
methylated and trimethylsilylated, then were subjected to GC–MS. TriHome: the reference compound 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid; PXG + 9-HPOD
(acid-hydrolysis): 9(S)-HPOD was incubated with SlPXG at 30 °C for 30 min, then the reaction solution was acidified to pH 3 and kept at 23 °C for 5 min. Numbers 6a, 7a, 10a
and 11a referring to trihydroxyoctadecenoic acid isomers correspond to that previously reported (Hamberg, 1991a; Hamberg and Hamberg, 1996). Peak V (this work) was
identified as 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid (according to Hamberg and Hamberg, 1996). Peaks VI and VII were identified as 9(S),12(R),13(R)-
trihydroxy-10(E)-octadecenoic acid (9b) and 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (10a), respectively (according to Hamberg, 1991a). (B) Metabolism of 9(S)-
HPOD by PXG and EH. R: (CH2)7-COOH; 9(S)-HPOD: 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid; 12(R),13(S)-epoxy-9(S)-HOD: 12(R),13(S)-epoxy-9(S)-hydroxy-
10(E)-octadecenoic acid; PXG: peroxygenase; EH: epoxide hydrolase.
The oxidation of linoleic acid by SlPXG yielded two positional
monoepoxide isomers, namely cis-12,13-epoxy-9(Z)-octadeceno-
ate (I) and cis-9,10-epoxy-12(Z)-octadecenoate (II) (Fig. 2A and E)
and corresponds to that of a previous study (Blée and Schuber,
1990). cis-12,13-Epoxy-9(Z)-octadecenoate and cis-9,10-epoxy-
12(Z)-octadecenoate were further converted into 12,13-dihy-
droxy-9(Z)-octadecenic acid (III) and 9,10-dihydroxy-12(Z)-octa-
decenic acid (IV), respectively, by NbEH2.1 and StEH (Fig. 2C–E).
However, no diol products were detected in the reaction contain-
ing NbEH1.1 (Fig. 2B).
It has been reported that 12(R),13(S)-epoxy-9(S)-hydroxy-
10(E)-octadecenoic acid was formed by incubation of 9(S)-HPOD
with PXG from broad bean (Hamberg and Hamberg, 1990, 1996).
In our study, we also observed only one compound when 9(S)-
HPOD was incubated with SlPXG. The major product was tenta-
tively identified as 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadec-
enoic acid (V, Fig. 3). The Me3Si derivative of compound V showed
a mass spectrum with prominent ions at m/z 383 (M-15, loss of
CH3), 241 [M-157, loss of (CH2)7-COOCH3], and 99 [+O„C–
(CH2)4–CH3], which were identical with those of the Me3Si deriva-

F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
11
tive of 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid
(Hamberg and Hamberg, 1996). The methyl esters of
9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (Fig. 3A, first
trace) and the PXG product 12(R),13(S)-epoxy-9(S)-hydroxy-
10(E)-octadecenoic acid which was acid-hydrolysed (Fig. 3A, sec-
ond trace) as described previously (Hamberg, 1991a; Hamberg
and Hamberg, 1996) were used as references. According to previ-
ous studies (Hamberg, 1991a,b; Hamberg and Hamberg, 1996),
products VI and VII (Fig. 3A) were identified as 9(S),12(R),13(R)-
and 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (Fig. 3B).
The result indicated that 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-
octadecenoic acid was converted into two isomers of trihydroxyoc-
tadecenoic acid, namely 9(S),12(S),13(S)- and 9(S),12(R),13(R)-tri-
hydroxy-10(E)-octadecenoic acid, by NbEH2.1 and StEH (Fig. 3A
and B). The result also revealed that StEH was much more active
towards 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid
than NbEH2.1. The ratio of 9(S),12(S),13(S): 9(S),12(R),13(R) iso-
mers produced by NbEH2.1 was 58: 42, but was 36: 64 when pro-
duced by StEH. A small amount of trihydroxyoctadecenoic acid was
also detected in the reactions containing only PXG protein or PXG
in combination with NbEH1.1 protein. These trihydroxyoctadece-
noic acids are probably derived directly from chemical hydrolysis
of compound V. Recently, we demonstrated by incubation of
12,13-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid in the pres-
ence of labeled H2O18 with StEH (Huang and Schwab, 2012) that
O18 is transferred to C12 and C13 of the fatty acid (ratio 1:ꢁ2.5).
This observation clearly confirms the formation of two trihydroxy
stereoisomers by StEH.
back inhibition was excluded and protein - protein interaction
between PXG and EH is assumed. To challenge this hypothesis, var-
ious amounts of purified NbEH1.1 as well as boiled NbEH1.1 (at
99 °C for 15 min, the enzyme was completely inactivated) were
co-incubation with SlPXG and linoleic acid. The more NbEH1.1
was added, the less epoxide was formed (Fig. 4). Unexpected,
boiled NbEH1.1 protein also inhibited the activity of SlPXG
(Fig. 4). It indicated that the inhibition of the PXG enzyme activity
did not depend on the activity of EH protein. The primary EH pro-
tein structure is enough to inhibit the PXG enzyme activity. How-
ever, the inhibition may not happen in a plant cell, as PXG is an
integral membrane protein (Blée, 1998) and EH is a soluble protein
in plants (Kiyosue et al., 1994; Stapleton et al., 1994; Arahira et al.,
2000; Morisseau et al., 2000; Bellevik et al., 2002).
2.5. Expression profiles of NbEH1.1 and NbEH2.1
2.5.1. Expression of NbEH1.1 and NbEH2.1 in N. benthamiana treated
with TMV based viral vectors and wounding
To examine whether NbEH1.1 and NbEH2.1 expression levels
correlate with biotic and abiotic stress, total RNA was isolated from
infiltrated leaves of N. benthamiana at various times after infection
with tobacco mosaic virus (TMV)-based viral vectors. NbEH1.1
transcript levels increased 4 days post infiltration and continued
to increase throughout the time course (Fig. 5A). In contrast, no sig-
nificant induction of NbEH2.1 expression was observed in the infil-
trated plants. EH enzyme activity was analyzed in the leaves
treated with the viral vectors using phenyl glycidyl ether as a sub-
strate. The EH activity was increased throughout the time course.
The activity in infiltrated leaves (10 days post infiltration) was 7-
fold higher than that in untreated leaves. The effect of wounding
on NbEH1.1 and NbEH2.1 gene expression was also tested by infil-
tration with buffer (detailed description see Experimental proce-
dures). As a result, the level of NbEH1.1 transcripts increased
strongly up to 24 h after wounding and decreased afterwards.
However, wounding had no significant effect on NbEH2.1 transcript
levels (Fig. 5B). Changes in EH expression levels during longer peri-
ods after buffer infiltration were not observed.
2.4. Inhibition of SlPXG catalytic activity by EH enzyme
We observed a phenomenon that SlPXG enzyme activity was
inhibited by all three EH enzymes. Neither epoxide nor diol prod-
ucts were produced when SlPXG and any of the purified EHs were
simultaneously incubated with fatty acids. This phenomenon was
also observed in the mixture of SlPXG- and StEH-infiltrated tobacco
extracts (Huang and Schwab, 2012). The inhibition could be over-
come when fatty acids were incubated with SlPXG yeast extracts at
first for at least 30 min, then, EH proteins were added to the reac-
tion. As no fatty acid diol was formed by NbEH1.1, product feed-
The importance of epoxide hydrolase during pathogen attack
may be related to its roles in detoxification, signaling, or
6
a
Epoxide
Linoleic acid
4
2
0
6
b
4
2
0
6
4
2
c
0
6
4
2
0
6
4
2
0
d
e
18
19
20
21
22
23
24
25
26
27
Time (min)
Fig. 4. Inhibition of PXG catalytic activity by NbEH1.1 protein. (a) SlPXG was incubated with linoleic acid. (b) NbEH1.1 (1
NbEH1.1 (2.5 g) was simultaneously incubated with (a). (d) NbEH1.1 (7.5 g, boiled at 99 °C for 15 min) was simultaneously incubated with (a). (e) NbEH1.1 (7.5
simultaneously incubated with (a).
l
g) was simultaneously incubated with (a). (c)
l
l
lg) was

12
F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
2.5.2. Spatial distribution of NbEH1.1 and NbEH2.1 gene transcripts in
the N. benthamiana plant
A
300
250
200
150
100
50
EHs have been localized in many cell types including seedlings,
germinated seeds, roots, fruit, tubers and leaves. The expression
patterns of NbEH1.1 and NbEH2.1 were also examined in various
N. benthamiana organs by real-time PCR (Fig. 5C). The gene tran-
scripts of NbEH1.1 were present in much higher levels in roots than
in flowers, sepals, leaves, and stems. The expression level in roots
was 19-fold higher than the lowest level found in flowers. As the
root system represents the first line of respond to pathogen attack
and water stress in plants, it is not surprising that NbEH1.1 was
only expressed in roots of unstressed plants, and was induced after
treatment of pathogen and wounding. Likewise, the transcript of
rough lemon RlemEH was not detected in flowers, fruits, stems,
or leaves, but was induced after inoculation of pathogen, wound-
ing, or treatment of methyl jasmonate (Gomi et al., 2003). NbEH2.1
gene transcripts were present predominantly in leaves and stems,
with a slightly higher level in young leaves. Also, the lowest level of
NbEH2.1 gene transcripts was found in flowers. A EH gene from
Arabidopsis (AtEH) whose amino acid sequence is 59% identical to
NbEH2.1 was also detected in the stems and leaves, but not in roots
and flowers (Kiyosue et al., 1994). The expression level of the pota-
to StEH was also found to be higher in meristematic tissue than in
mature leaves (Stapleton et al., 1994).
0
UT
4d
7d
10d
B
C
20
18
16
14
12
10
8
6
4
2
0
UT
0.5h
1h
2h
6h
24h
48h
3. Conclusion
35
30
25
20
15
10
5
In order to verify similarities and differences in the roles be-
tween EH1 and EH2 groups in plants, we have analysed the cata-
lytic functions and gene expression patterns of NbEH1.1 and
NbEH2.1 from N. benthamiana. The enzymes differed in substrate
specificity and in contrast to NbEH1.1, NbEH2.1 displayed a broad
substrate range. These differences may be due to differences in the
lid region around the catalytic site of the EHs (Wijekoon et al.,
2011). Moreover, we showed that NbEH2.1, in contrast to NbEH1.1
was active towards all fatty acid epoxides examined, similar to
StEH. This result coincided with the sequence analysis which re-
vealed that EH2 enzymes have a predicted peroxisome targeting
signal type 1 (PTS1) C-terminus (Neuberger et al., 2003). The main
functions of peroxisomes are defence against oxidative stress and
b-oxidation of fatty acids (Nyathi and Baker, 2006). It is suggested
that the EH2 group may be associated with peroxisomes and that
they are involved in cutin monomer production in plants (Blée
and Schuber, 1993).
The peroxygenase pathway has been perceived to be involved in
the biosynthesis of oxylipins in response to environmental stress
or in the biosynthesis of cutin polymers that are major constituents
of plant surfaces (Blée, 1998). In this study, we reconstituted the
peroxygenase pathway in vitro using a mixture of SlPXG yeast ex-
tract and purified recombinant EH proteins. Only the incubation of
the mixture of SlPXG and EH2 proteins with oleic acid, cis-11-
eicosenoic acid, linoleic acid, and 9(S)-HPOD produced a high level
of diols or triols. This supported the suggestion that EH2 but not
EH1 are involved in cutin monomer production in plants. Our data
also showed differential gene expression levels of NbEH1.1 and
NbEH2.1 in response to pathogen and wounding as well as in var-
ious organs in N. benthamiana. Thus, they are differently regulated
and may serve diverse roles.
0
Flower Sepal mature young Stem Root
leaf leaf
Fig. 5. Quantitative real-time RT-PCR analysis. (A) The effect of tobacco mosaic
virus (TMV) on gene expression levels of NbEH1.1 and NbEH2.1 in N. benthamiana.
Leaves were infiltrated with TMV based viral vectors and harvested at 4, 7, and
10 days after infiltration (4d, 7d and 10d). UT: untreated tobacco leaves. The
expression level of NbEH1.1 in untreated tobacco leaves was defined as 1. (B) The
effect of wounding on gene expressions of NbEH1.1 and NbEH2.1 in N. benthamiana.
Leaves were infiltrated with buffer and were harvested at 30 min, 1 h, 2 h, 6 h, 24 h,
and 48 h after wounding treatment. UT: untreated tobacco leaves. The expression
level of NbEH1.1 in untreated tobacco leaves was defined as 1. (C) Spatial
distribution of NbEH1.1 and NbEH2.1 gene transcripts in N. benthamiana plant.
The expression level of NbEH1.1 in flowers was defined as 1. Quantitative real-time
RT-PCR analysis was performed using NbEH1.1, NbEH2.1 and 18S-26S interspacer
gene specific primers, the latter used as internal control for normalization. Values
are means SEM of three different evaluations carried out with two sets of cDNAs.
metabolism of antimicrobial compounds. Previous studies showed
that NtEH1 from Nicotiana tabacum was induced during the hyper-
sensitive response to TMV (Guo et al., 1998) and NbEH1.1, NbEH1.2,
NbEH2.1 and NbEH2.2 respond differently to various pathogens
(Wijekoon et al., 2008, 2011). The results implied specialization
among EH genes in basal resistance. In this study, we also demon-
strated differential gene expression of NbEH1 and NbEH2 due to
wounding and pathogen infiltration. Similarly, the transcripts of
StEH accumulated upon wounding and application of exogenous
methyl jasmonate (Stapleton et al., 1994). Other plant hormones
such as auxin and ethylene have been reported to induce the
expression of AtEH and GmEH, respectively (Kiyosue et al., 1994;
Arahira et al., 2000).
Overall, different isoforms of EHs probably exhibit different bio-
chemical and biological functions in plants as they accept different
epoxide substrates and show various expression patterns. Our re-
sults showed that NbEH1.1 only accepted aromatic epoxides such
as phenyl glycidyl ether and styrene oxide as substrates. As
NbEH1.1 was induced by TMV and wounding (this study) and
had increased expression during compatible interactions with

F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
13
some pathogens (Wijekoon et al., 2008), it may play a role in gen-
erating signals for activation of certain defence responses in tobac-
co. However, EH2 enzymes which contain a predicted peroxisomal
targeting sequence at the C terminus, hydrate efficiently fatty acid
epoxides to form diols and are uniformly expressed in different tis-
sues may be associated with peroxisomes and are probably in-
volved in cutin monomer production in plants.
number U57350), a tobacco NbEH2 gene (Genbank accession num-
ber EU779658), and a potato EH gene (Genbank accession number
U02497), respectively. The PCR primers used for each gene are
listed in Supplementary Table S2. The temperature program used
was 5 min at 95 °C, 1 cycle; 45 s at 95 °C, 45 s at 55 °C, 2 min at
72 °C, 35 cycles; final extension at 72 °C for 10 min. The PCR prod-
ucts were double digested with restriction enzymes, the recogni-
tion sequences (underlined) of which are contained in the
primers (Supplementary Table S2), and then ligated with the
pQE30 vector (QIAGEN, Hilden, Germany) to yield pQE30-NbEH1.1,
pQE30-NbEH2.1 and pQE30-StEH. The recombinant genes were
subjected to sequencing to confirm the sequence of the inserts.
Furthermore, these three constructs and empty vector (negative
control) were transformed into the M15 (pREP4) cell strain for
expression of recombinant protein. A 4 ml overnight culture was
used to inoculate a 400 ml culture in LB medium containing
4. Experimental
4.1. Chemicals
Commercial chemicals were purchased in analytical grade from
the following companies: linoleic acid (Roth, Karlsruhe, Germany);
oleic acid, cis-11-eicosenoic acid, cis-stilbene oxide, trans-stilbene
oxide, trans-1,3-diphenyl-2,3-epoxypropan-1-one, phenyl glycidyl
ether, styrene oxide, 1,2-epoxypentane, and glycidyl isopropyl
ether (Sigma-Aldrich, Steinheim, Germany); rac cis-9,10-epoxyste-
aric acid (Biozol Diagnostica, Eching, Germany). 9(S)-HPOD was
synthesized from linoleic acid using Nb-9-LOX from N. benthami-
ana (Huang and Schwab, 2011).
100 l l
g mlꢀ1 ampicillin and 25 g mlꢀ1 kanamycin. Cultures were
grown at 37 °C until an OD600 of 0.6 was reached. Expression of
the protein was induced by the addition of 0.2 mM IPTG, and the
cultures were grown at 18 °C for an additional 20 h. Cells were har-
vested by centrifugation (5000g, 20 min, 4 °C) and resuspended in
10 ml binding buffer (20 mM sodium phosphate buffer, 500 mM
NaCl, 20 mM imidazole, pH 7.4). Cells were lysed by sonication
on ice with a MS 73 sonotrode (Bandelin electronic, Berlin Ger-
many) 10 times for 30 s at 10% of maximal power. Cell debris
was removed by centrifugation (12,000g, 30 min, 4 °C). Purification
of the expression products of NbEH1.1, NbEH2.1 and StEH were per-
formed using a His trap affinity column (GE Healthcare, Freiburg,
Germany), prepacked with precharged Ni Sepharose 6 fast flow,
according to manufacturer’s instructions. Proteins were analyzed
by SDS–PAGE with 10% polyacrylamide gels and stained with Coo-
massie brilliant blue R-250. The protein concentration was deter-
mined by the Bradford assay (Bradford, 1976).
4.2. Treatment of plant materials
N. benthamiana was grown in a growth room maintained at
23 1 °C with a 16 h light/8 h dark photoperiod and a light inten-
sity of 70 10 l
mol mꢀ2 sꢀ1. Leaves used for treatment of wound-
ing or infiltration of the viral vectors were taken from plants about
7–10 weeks old. For investigation of the effects of wounding on
NbEH1.1 and NbEH2.1 gene expression, leaves of N. benthamiana
were infiltrated with buffer (10 mM 2-N-morpholino-ethanesulfo-
nic acid (MES) pH 5.5, 10 mM MgSO4) and were harvested at
30 min, 1 h, 2 h, 6 h, 24 h, and 48 h after wounding. The description
of the viral vectors and agroinfiltration of tobacco leaves were pre-
viously reported (Huang and Schwab, 2011). The tobacco leaves
harvested 4, 7, and 10 days after infiltration were used for real time
PCR analysis.
4.5. Enzyme assays
EH activities towards various substrates were determined by
LC–MS and GC–MS as described below.
For aromatic epoxide substrates (Supplementary Fig. S1A), EH
activity was determined by LC–MS. Purified proteins were incu-
4.3. Real-time RT-PCR analysis
bated with 300
l
M substrate in 500
l
l of 1ꢂ PBS buffer (140 mM
Total RNA was extracted from N. benthamiana using the CTAB
extraction procedure (Liao et al., 2004). RNA samples were treated
with RNase free DNase I (Fermentas, St. Leon-Roth, Germany) for
1 h at 37 °C. First strand cDNA synthesis was performed in dupli-
NaCl, 8 mM Na2HPO4, 2.7 mM KCl, 1.76 mM KH2PO4, pH 7.4) for
30 min at 30 °C. The reaction products were extracted with the
same volume of ethyl acetate, evaporated to dryness, resuspended
in methanol, and analysed by LC–MS (method I, see below).
For terminal aliphatic epoxide (Supplementary Fig. S1B) and
limonene epoxide (Supplementary Fig. S1C) substrates, EH activity
was determined by SPME-GC–MS. Purified proteins were diluted to
cate in a 20 ll reaction volume, with 1 lg of total RNA as the tem-
plate, random primer (random hexamer, 100 pmol), and M-MLV
reverse transcriptase (200 U, Invitrogen, Karlsruhe, Germany)
according to the manufacturer’s instructions. Real-time PCR was
performed as described by Huang et al. (2010). Relative quantifica-
tion of gene expression was performed using an 18S–26S intersp-
acer gene as a reference (Pfaffl, 2001). Primers used are listed in
Supplementary Table S1.
2 ml of 1ꢂ PBS buffer containing 300
lM substrate. The mixture
was incubated for 30 min at 30 °C with constant shaking in a
20 ml reaction vial closed with a septum. Headspace compounds
were trapped by SPME (65 lm polydimethylsiloxane–divinylben-
zene coated fibre, Supelco, Steinheim, Germany) at 45 °C for
30 min and analysed by GC–MS (method I for terminal aliphatic
epoxide substrates and method II for limonene epoxide substrates,
see below).
4.4. Heterologous expression and purification of NbEH1.1, NbEH2.1
and StEH
Fatty acid epoxides (Supplementary Fig. S1D, except for cis-
9,10-epoxystearic acid) were prepared by incubation of corre-
sponding fatty acids with SlPXG (peroxygenase from tomato) yeast
extract (Aghofack-Nguemezi et al., 2011) at 30 °C in buffer C
(10 mM sodium acetate buffer, pH 6.0, 2% glycerol, 0.01% Tween
Total RNA was isolated from leaves of N. benthamiana treated
with viral vectors and potato tuber (Solanum tuberosum) by CTAB
extraction (Liao et al., 2004). The first-strand cDNAs were
synthesized from 10 lg of total RNA using Superscript III RTase
(Invitrogen, Karlsruhe, Germany) and a GeneRacer oligo-dT primer
(50-GCT GTC AAC GAT ACG CTA CGT AAC GGC ATG ACA GTG T(18)-30).
The coding regions of NbEH1.1, NbEH2.1, and StEH were ampli-
fied by RT-PCR with the corresponding cDNA template and primers
designed on the basis of a tobacco NtEH gene (Genbank accession
20) containing 2.5 mM hydrogen peroxide and 300 lM of fatty acid
with constant shaking for 60 min. The PXG products were parti-
tioned into ethyl acetate, evaporated to dryness, and further used
as substrates for EH enzyme assays. Purified EH proteins were
incubated with various fatty acid epoxides in excess in 500 ll of

14
F.-C. Huang, W. Schwab / Phytochemistry 90 (2013) 6–15
1ꢂ PBS buffer for 30 min at 30 °C. The reaction products were ex-
tracted with the same volume of ethyl acetate, evaporated to dry-
ness, re-suspended in methanol, and analysed by LC–MS (method
II, see below). Products formed from 9(S)-HPOD with SlPXG in
combination with each EH were isolated by extraction with ethyl
acetate, methylated and trimethylsilylated, and then subjected to
GC–MS (Aghofack-Nguemezi et al., 2011).
Acknowledgements
Financial Support by the German Federal Ministry of Nutrition,
Agriculture and Consumer Protection (BMELV) and FNR (SynRg) is
gratefully
acknowledged.
The
reference
compound
9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid was kindly
provided by Mats Hamberg.
Amounts of aromatic diol products were determined using a
standard curve calculated from various known concentrations of
corresponding substrates against the UV peak areas which were re-
corded at 254 nm. Amounts of fatty acid diol products were deter-
mined using a standard curve calculated from various known
concentrations of cis-9,10-epoxystearic acid (Biozol, Diagnostica,
Eching, Germany) against the mass peak areas which were re-
corded by LC–MS. Amounts of trihydroxyoctadecenoic acid prod-
ucts were determined using a standard curve calculated from
various known concentrations of methylated and trimethylsilylat-
ed 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid against the
mass peak areas which were recorded by GC–MS.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
02.020.
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The pH optimum of NbEH1.1 and NbEH2.1 was determined
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(pH 2.0–6.0), 50 mM sodium phosphate (pH 6.0–8.0), and 50 mM
Tris–HCl (pH 8.0–12.0) in 0.5-U intervals. The optimal temperature
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The HPLC system consisted of a quaternary pump and a variable
wavelength detector, all from Agilent 1100 (Bruker Daltonics, Bre-
men, Germany). The column was a LUNA C18 100A 150 ꢂ 2 mm
(Phenomenex, Aschaffenburg, Germany). HPLC was performed
with the following binary gradient system: solvent A, water with
0.1% formic acid and solvent B, 100% methanol with 0.1% formic
acid. Two different gradient programs were used: Method I: 0–
10 min, 50% A/50% B to 100% B, hold for 5 min; 15–20 min, 100%
B to 50% A/50% B, hold for 10 min; method II: 0–10 min, 70% A/
30% B to 50% A/50% B; 10–40 min, 50% A/50% B to 100% B, hold
for 5 min; 100% B to 70% A/30% B, in 1 min, then hold for 6 min.
The flow rate was 0.2 ml minꢀ1. Attached to the HPLC was a Bruker
esquire 3000plus mass spectrometer with an ESI interface that was
used to record the mass spectra. The ionization voltage of the cap-
illary was 4000 V and the end plate was set to ꢀ500 V. Products
were monitored using diagnostic ions listed in Supplementary
Table S3.
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