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Mycopathologia 140: 51–58, 1997.
© 1997 Kluwer Academic Publishers. Printed in the Netherlands. 51
Characterization of epoxide hydrolase activity inAlternaria alternata f. sp.
lycopersici. Possible involvement in toxin production
Epoxide hydrolase in Alternaria alternata f. sp. lycopersici
Franck Pinot1,3, Eloisa D. Caldas2, Christina Schmidt3, David G. Gilchrist4, A.D. Jones5,Carl
K. Winter6& Bruce D. Hammock3
1Laboratoire d’Enzymologie Cellulaire et Mol´eculaire, Institut de Biologie Mol´eculaire des Plantes, Strasbourg,
France;
2Health Institute of Federal District, Brasilia, DF, Brazil;
3Departments of Entomology and Environmental Toxicology;
4Plant Pathology Department;
5Facility for Advanced Instrumentation;
6Department of Food Science and Technology, University of California, Davis, California
Received 8 September 1997; accepted 16 December 1997
Abstract
Using trans-diphenylpropane oxide (tDPPO) as a substrate, we measured epoxide hydrolase (EH) activity in sub-
cellular fractions of Alternaria alternata f. sp. lycopersici (Aal), a fungus that produces host-specific toxins. The
activity was mainly (>99.5%) located in the soluble fraction (100,000 ×g supernatant) with the optimum pH at
7.4. An increase of toxin production between days 3 and 9 found in a Aal liquid culture over a 15 days period
was concomitant with a period of high EH activity. EH activity remained constant during the same period in an
Alternaria alternata culture, a fungus which does not produce toxin. In vivo treatment of Aal culture with the
peroxisome proliferator clofibrate stimulated EH activity by 83% and enhanced toxin production 6.3 fold. Both 4-
fluorochalcone oxide (4-FCO) and (2S,3S)-(-)-3-(4-nitrophenyl)-glycidol (SS-NPG) inhibited EH activity in vitro
with a I50 of 23 ±1µM and 72 ±19 µM, respectively. The possible physiological substrate 9,10-epoxystearic
acid was hydrolyzed more efficiently by Aal sEH than the model substrates trans- and cis-stilbene oxide (TSO and
CSO) and trans-andcis-diphenylpropane oxide (tDPPO and cDPPO).
Key words: Epoxide hydrolase, fungus, inhibitor, AAL toxins
Abbreviations:CSO,cis-stilbene oxide; cDPPO, cis-diphenylpropene oxide; EH, epoxide hydrolase; 4-FCO,
4-fluorochalcone oxide; oxide; SS-NPG, (2S, 3S)-(-)-3-(4-nitrophenyl)-glycidol; tDPPO, trans-diphenylpropene
oxide; TSO, trans-stilbene oxide.
Introduction
Epoxide hydrolases (EHs) (EC 3.3.2.3) convert epox-
ides to diols by addition of water. The cloning of EHs
from different origins enabled authors [1, 2] to per-
form sequence homology analysis and classify them
in the group of enzymes known as the α/βhydrolase
fold family [3]. The broad spectrum of the mam-
malian soluble EH (sEH), in addition to the fact that
hepatic microsomal EH (mEH) is induced by a vari-
ety of foreign compounds, suggests their involvement
in xenobiotic metabolism [4, 5]. The tissue specific
regulation of sEH in mice might indicate its different
physiological roles [6], including participation in the
metabolism of endogenous compounds, since epox-
ides of fatty acids are hydrolyzed more rapidly than
many other substrates [7].
Croteau and Kolattukudy [8] were the first au-
thors to report the presence of EH in a plant, which
was located in a 3,000 ×g particulate fraction from
52
homogenates of apple skin. A sEH has also been
purified from soybean [9] and both enzymes have
been proposed to participate in synthesis of cutin
monomers. Indeed, they are able to produce diols of
fatty acids which are incorporated in plant envelopes
[10]. More recently, inducible EHs have been cloned
from mouse eared cress (Arabidopsis thaliana)and
potato (Solanum tuberosum) [11, 12].
EHs have also been detected in fungi. Kolattukudy
and Brown [13] partially purified an EH from Fusar-
ium solani pisi and fungal EHs have been used in
organic synthesis for enantioselective hydrolysis [14–
17]. However, little information is available on these
enzymes and their physiological significance remains
to be established. They might be important in plant-
fungus interaction. Diols of fatty acids induce a cuti-
nase of F. solani pisi, which facilitates the penetration
of the fungus in the plant [18]. Alternaria alternata
fsp.lycopersici (Aal), is a fungal pathogen which
causes the stem canker in tomato [19], a disease
elicited by the toxins produced by the fungus (AAL
toxins) [20]. Alternaria alternata (Aa) is a fungus
morphologically similar to Aal, but which does not
produce detectable toxin. The presence of two pairs
of vicinal diols free or esterified in the AAL toxins
(Figure 1) suggests the possible involvementof EH in
their synthesis.
The presence of EH activity in Alternaria alternata
fsp.lycopersici, its substrate specificity and the ef-
fect of inhibitors on the activity in vitro are described
in this paper. In an attempt to understand the role of
EH in this fungus, the correlation between toxin pro-
duction and EH activity in both Aal and Aa liquid
cultures and the effect of in vivo treatment of an EH
inducer (clofibrate) on both Aal EH activity and toxin
production were evaluated.
Materials and methods
Chemicals. KCN, Na2B4O7.10H2O, KH2PO4and
ethyl 2-(4-chlorophenoxy)-2-methylpropionate (clofi-
brate) were purchased from Aldrich Chemical Com-
pany (Milwaukee, WI). 3-(4-Nitrophenyl)glycidol
(SS-NPG and RR-NPG) and BSA were purchased
from Sigma (St. Louis, MO). Naphthalene-2,3-
dicarboxaldehyde (NDA) was obtained from Mole-
cular Probes, Inc (Eugene, OR). 4-Fluorochalcone
oxide (4-FCO) was synthesized as reported previously
[21]. [2-3H]-trans-1,3-Diphenylpropeneoxideand[2-
3H]-cis-1,3-Diphenyl propene oxide ([3H]-tDPPO and
[3H]-cDPPO) were synthesized according to Borhan
et al. [22]. [2-3H]-trans-Stilbene oxide and [2-3R]-
cis-stilbene oxide ([3H]-TSO and [3H]-CSO) were
synthesized according to Mullin and Hammock [23].
[1-14C]-9,10-Epoxystearic acid was synthesized from
[14-C]-oleic acid (DuPont-New England Nuclear,
Boston, MA, USA) using m-chloroperoxybenzoic
acid. [1-14C]- Methyl-9,10-epoxystearate was syn-
thesized by methylation of [1-14C]-9,10-epoxystearic
with diazomethane. All solvents used were HPLC
grade (Fisher Scientific).
Subcellular fractionation. Fungal mycelium (2–5
grams) was homogenized (Polytron, Brinkmann In-
struments, Westbury, NY) in 0.1 M phosphate buffer
(pH 7.4) containing 250 mM sucrose, 1 mM EDTA,
15 mM β-mercaptoethanol and 1 mM phenylmethyl-
sulfonylfluoride. The homogenate was filtered through
cheesecloth and centrifuged for 25 min at 10,000 ×g
at 4◦C. The pellet was resuspended in 0.1 M phosphate
buffer (pH 7.4), aliquoted and stored at -80◦C. A part
of the supernatant was aliquoted and stored at −80◦C,
the rest was centrifuged at 100,000 ×g for 65 min.
The supernatant (soluble fraction) was aliquoted and
stored at −80◦C, the pellet (microsomes) was resus-
pended in 0.1 M phosphate buffer (pH 7.4) containing
30% glycerol and stored at −80◦C. The protein con-
centration of the samples was determined with the
Biorad protein assay kit (Bio-Rad, Hercules, CA)
using bovine serum albumin as a standard.
Determination of EH activity. EH activity was mea-
sured with 10,000 g supernatant (5 µg of protein)
using radiolabeled tDPPO as already described [22],
after incubation for 10 min at 27◦C. For the subcel-
lular location of the activity, the amount of protein
used per incubation was 5 µg for crude extract and
10,000 g supernatant and 68, 8 and 660µg for 10,000
g pellet, soluble fraction and microsomes, respec-
tively. Optimum pH was determined after incubation
of protein with 100 µM of tDPPO in phosphate buffer
(pH 4.5–7.4) and tris buffer (pH 7.8–9.3), with a
0.1 M ionic strength. For the substrate specificity
study, EH activity was measured using radiolabeled
substrates according to Pinot et al. [24] with 9,10-
epoxystearic and methyl-9,10-epoxystearate, Mullin
and Hammock [23] with TSO and CSO and Borhan
et al. [22] with tDPPO and cDPPO. All the assays
were run in 0.1 M phosphate buffer (pH 7.4) at 27◦C
for 10 min with a substrate concentration of 100 µM.
The amount of protein from the soluble fraction in
53
Figure 1. Structure of TA AAL toxins.
each incubation was: 5 µg (TSO, CSO, tDPPO), 12
µg (cDPPO), 16 µg (9,10-epoxystearic acid and the
corresponding methyl ester). All assays were shown
to be linear for both protein concentration and time.
For inhibition studies, the 10,000 g supernatant was
pre-incubated with concentration ranging from 1 to
50 µM for 4-FCO and from 10 to 150 µM for SS-
NPG and RR-NPG. Controls were pre-incubated with
solvent (ethanol) only. After 10 minutes, the substrate
(tDPPO) was added and the reaction was allowed to
proceed.
Fungal cultures. Alternaria alternata fsp.lycoper-
sici and Alternaria alternata were grown in a pectin
liquid medium [25] at 25◦C. The fungi were har-
vested at 2–15 days after inoculation, according to
each experiment. The mycelial suspension was filtered
in cheesecloth and squeezed to eliminate the excess
water. The mycelium was immediately prepared for
measurement of EH activity or kept at −80◦Cforfur-
ther analysis. EH activity was measured with 5 µg
of protein from the 10,000 ×g supernatant incubated
with 100 µM tDPPO for 10 min at 27◦C. An aliquot of
the liquid medium (2 ml) was filtered in 0.45 mmny-
lon filter and analyzed for toxin production (TA AAL
toxin) by the HPLC/NDA method. For induction stud-
ies, clofibrate (in ethanol at 1 mM final concentration)
was added to cultures which were2 or 3 days old and
the cultures harvested at day 6. Ethanol was added to
the controls.
Measurement of toxin production. KCN (300 µmol)
was added to a 3 ml vial contained 10 µl of sam-
ple (liquid medium), followed by 260 µLof0.05M
Na2B4O7. 10H20 (pH 8.5) and 150 µmol of NDA.
The sample was mixed thoroughly and the reaction
mixture incubated at 60◦C for 15 minutes in a Re-
actiTherm heating module (Pierce Chemical). The
analysis was performed using a HPLC Beckman Sys-
tem Gold system with Ultracarb 5 ODS 30 (4.5 ×10
mm, Phenomenex) column, coupled to a Perkin Elmer
650 Fluorescence Detector (Ex 420 nm, 10 nm slit,
Em 490 nm). The mobile phase consisted of 56% ace-
tonitrile (44% 50 mM H3PO4adjusted to pH 2.8 with
NH4OH). TA AAL toxin, purified in our laboratory
according to Caldas et al. [25] and shown to be at least
95% pure based on the 1H-NMR spectrum, was used
as standard.
Results and discussion.
EH activity in extracts from Aal grown in liquid cul-
ture was measured in different subcellular fractions
with tDPPO (Table 1). The highest specific activ-
ity was found in the 10,000 ×g supernatant. When
reported as total activity, the results show that af-
ter the 100,000 ×g centrifugation, the activity is
mainly (>99.5%) located in the soluble fraction. This
corroborates previous work performed with another
fungus, Fusarium solani pisi [13]. Mammalian EH
activity is also mostly located in the soluble fraction.
In a recent work, Borhan et al. [22] measured the
metabolism of five different substrates by four soluble
and three microsomal EHs from different organisms.
They found that the specific activity of the microsomal
EHs were at least two orders of magnitude lower than
the specific activity of the soluble enzymes for the sub-
strates examined. Except for the substrate specificity
study which was performed with the soluble frac-
tion (100,000 ×g), all activity measurement in this
work were done with the 10,000 ×g supernatant and
54
Figure 2. Effect of pH on sEH activity from Alternaria alternata fsp.lycopersici.
Table 1. Epoxide hydrolase activity in subcellular fractions from Al-
ternaria alternata fsp.lycopersici
Total activity Specific activity
Fraction nmol/min % (nmol/min/mg)
Crude extracts 490 100 3.92
10,000 ×g supernatant 479 98 5.71
10,000 ×g pellet 8 1.7 0.24
100,000 ×g supernatant 478 99.8 3.99
(soluble fraction)
100,000 ×g pellet 0.8 0.2 0.03
(microsomes)
the enzymatic system designated as soluble epoxide
hydrolase (sEH).
In order to determine optimum pH, specific activity
of sEH was measured at pH ranging from 4.5 to 9.3
(Figure 2). Hydrolysis of tDPPO was maximum at pH
7.4. Interestingly, this pH differsfrom the optimum pH
from Fusarium solani pisi (pH 9) [13] and is similar
to the optimum pH reported for plant and mammalian
sEH (approximately 7.4) [5, 9].
During the infection of tomato by Aal, the fungus
produces host specific toxins (AAL toxins) which are
aliphatic chains with two pairs of vicinal diols free or
esterified [25, 26] (Figure 1). EH activity and toxin
production were measured over a 15 days period in a
liquid culture of Alternaria alternata fsp.lycopersici
(Aal) and Alternaria alternata (Aa), a non toxin pro-
ducer. As shown in Figure 3, in Aal, there is a peak
of sEH activity at days 3 and 6, period in which toxin
production increases drastically. At day 9, when sEH
activity has already dropped, toxin production stabi-
lized. In Aa, EH activity remained constant during the
same period. The fact that there is a change in sEH
activity during the log phase of toxin productionin Aal
culture while EH activity remains practically constant
for the non toxin producer (A. alternata), suggests a
possible relationship between epoxide hydrolase ac-
tivity and toxin biosynthesis. sEH may be important
55
Figure 3. sEH activity and toxin production in liquid culture of Alternaria alternata fsp.Lycopersici (Aal) andAlternaria alternata (Aa).
in the early stage of toxin biosynthesis, as enzyme
activity increases before toxin production could be
detected.
A better understanding of the physiological role
of sEH in Aal may be gained by studying the ef-
fect of inducers and inhibitors. Aal was grown in
media containing 1 mM of clofibrate, a peroxisome
proliferator shown to induce mammalian sEH [27].
Clofibrate treatment stimulated sEH activity by 83%
(3.27 ±0.5 nmol/min/mg protein in controls and 6 ±
1.4 in treated samples). This concomitant induction
of sEH activity and stimulation of toxin production
is in favor of the sEH participation in biosynthesis
of toxins. However, the effect of this treatment was
more pronounced on the toxin production which was
enhanced 6.3 fold (9.4 ±1.0 nmol/ml in controls and
59.0 ±9.1 nmol/ml in treated samples). The mech-
anism of action of this peroxisome proliferator is still
unclear, however it has been proposed that it occurs via
the activation of a group of transcription factors, the
peroxisome proliferators activated receptors (PPAR)
[28]. It is known that peroxisomeproliferators cause a
wide range of physiological effects [27]. Enhancement
of toxin production certainly could result from many
events possibly unrelated to the induction of sEH.
SS-NPG and 4-FCO have been shown to inhibit
sEHs, but not mEH, from different organisms[11, 12,
21, 29]. We studied the effect of these compounds by
measuring hydrolysis of tDPPO after pre-incubation
of the 10,000 ×g supernatant of Aal extracts with
different concentrations of RR-NPG, SS-NPG and 4-
FCO. The latter two compounds inhibited sEH activity
and inhibition increased with concentration (Figure 4).
I50 of 23 ±1µM and 72 ±19 µM for 4-FCO and
SS-NPG, respectively, were determined from these ex-
periments. No alteration of activity was observed with
the 1R,3R isomer of NPG, even at a concentration of
300 µM (data not shown). Studies performed with
sEHs from mammals and plants [11, 12, 29] have
shown that RR-NPG is a much less potent inhibitor
than the SS enantiomer. The mechanism of the inhibi-
tion of EH by these compounds is still unknown, but
according to the catalytic mechanism of EHs [30, 31]
it is assumed that these inhibitors act as tight binding
substrates.
Substrate selectivity in Aal sEH was investigated
by measuring the hydrolysis of different substrates by
the soluble fraction (100,000 ×g). As shown in Table
2, trans-stilbene oxide (TSO), a model substrate for
studies of mammalian sEH, was hydrolyzed twice as
fast as its cis isomer (CSO). tDPPO and cDPPO, which
56
Figure 4. Inhibition of sEH activity fromAlternaria alternata fsp.lycopersici by SS-NPG and by 4-FCO. Results are expressed as percent of
activity measured after pre-incubation withsolvent only: 6.9±0.6 nmol/min/mg protein (SS-NPG) or 6.5 ±0.8 nmol/min/mg protein (4-FCO).
Data are mean of three independent measurements performed in triplicate.
have been recently developed as surrogate substrates
in the laboratory [22], differ from TSO and CSO by
the presence of an additional methylene group. The
presence of this methylene resulted in an increase of
specific activity by one order of magnitude and by a
loss of selectivity for the trans and cis isomer. The
highest activity was measured with the physiological
substrate 9,10-epoxystearic acid. The methyl ester of
9,10-epoxystearic acid was hydrolyzed at a 10-fold
lower rate than the free acid. This observation indi-
cates that a free carboxyl group might be important
for the positioning of the substrate in the active site.
In contrast, however, Borhan et al. [22] showed
that all mammalian sEHs tested hydrolyzed tDPPO
between 3.8 and 15.2 times faster than 9,10-
epoxystearic acid. It has been shown that 9,10,18-
trihydroxystearic acid, which results from the hydrol-
ysis of 18-hydroxy-9,10-epoxystearic acid, induces
cutinase, an enzyme that facilitates the penetration
of the fungus in the plant [18]. Diols of fatty acids
have antimicrobial activities [32, 33, 34]. The fact
that the fungal sEH described here hydrolyses prefer-
entially the physiological substrate 9,10-epoxystearic
acid suggests that this enzyme might have a key role
in the process of plant infection. Aal could, via pro-
duction of diols, use sEH in interactions (i.e. compe-
tition) with other microorganisms. Fungi, like other
organisms, are in continuous contact with epoxides,
which are ubiquitous and occur naturally in industrial
settings, in the environment and in biochemical path-
ways. Some of them are highly toxic [35]. In contrast
to animals, fungi do not have an effective excretion
pathway and sEH would be the ideal enzyme to metab-
olize such reactive compounds, including a possible
intermediate in the AAL toxin biosynthesis.
We are now in the process of purifying the enzyme.
Working with pure enzyme will facilitate metabolic
studies and will help to get a better understanding of
its significance. The purification will also lead to a
molecular biology work. Cloning of the enzyme will
57
Table 2. Specific activity of sEH Alternaria alternata f. sp. lycopersici with dif-
ferent substrates. Results are mean of three independent experiments performed in
triplicate
Substrate Specific activity (nmol/min/mg)
indicate how the fungal EH that we studied is related
to EHs previously cloned from other organisms [22,
30].
Acknowledgements
This work was supported by Grant 2 RO1 E502710
from NIEHS and by Grant PHS RO1 HD 28253. U.C.
Davis is an NIEHS EnvironmentalHealth Center (C1
P30 ES 05707) and an EPA Center for Ecological
Health Research (CR819658).E. D. C. was supported
by a scholarship from the Brazilian Government.
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Address for correspondence: Professor Bruce D. Hammock, De-
partments of Entomology and Environmental Toxicology, Univer-
sity of California, 95616 Davis, California.
Tel: 916-752-7519; Fax: 916-752-1537; E-mail: bdham-
mock@ucdavis.edu.
