Purification and partial amino acid sequences of an esterase from tomato

Article abstract of DOI:10.1016/S0031-9422(02)00126-7

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M_r of 26 kDa (gel-filtration chromatography), which was similar to the M_r determined by SDS-PAGE and MALDI-TOF analysis (M_r of 28547 kDa). Enzyme kinetics revealed a K_m value of 15 μM and a V_max value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 °C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the α/β hydrolase fold protein superfamily.

Full text of DOI:10.1016/S0031-9422(02)00126-7

Phytochemistry 60 (2002) 233–240
www.elsevier.com/locate/phytochem
Purification and partial amino acid sequences of
an esterase from tomato
Christiane Stuhlfeldera, Friedrich Lottspeichb, Martin J. Muellera,*
aJulius-von-Sachs-Institute for Biosciences, Pharmaceutical Biology, University of Wurzburg, Julius-von-Sachs-Platz 2,
¨
D-97082 Wurzburg, Germany
¨
bMax-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany
Received 10 January 2002; received in revised form 25 March 2002
Abstract
Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing
enzyme activity (0.21–5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified
from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration
and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of
2.2%. The native enzyme exhibited a Mr of 26 kDa (gel-filtration chromatography), which was similar to the Mr determined by
SDS-PAGE and MALDI-TOF analysis (Mr of 28547 kDa). Enzyme kinetics revealed a Km value of 15 mM and a Vmax value of 7.97
nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 ꢀC. The enzyme also efficiently hydrolyzed methyl esters of
abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were
only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and
carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a
concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endo-
proteinase LysC revealed three peptide fragments with 11–14 amino acids. N-Terminal sequencing yielded an additional peptide
fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and
hydroxynitrile lyases that belong to the a/b hydrolase fold protein superfamily. # 2002 Published by Elsevier Science Ltd.
Keywords: Lycopersicon esculentum; Solanaceae; Protein purification; Methyl jasmonate; Jasmonic acid
1. Introduction
exogenously to plant cells, accumulation of defence
proteins and antimicrobial secondary metabolites
(Mueller et al., 1993) is observed. A model was pro-
posed in which receptor activation by the endogenous
peptide systemin, a systemic wound signal, or pathogen
derived signals leads to liberation of linolenic acid from
membrane lipids (Farmer and Ryan, 1992; Ryan, 2000).
Linolenate is rapidly converted by constitutive enzymes
to JA that directly or indirectly activates defence genes
(Mueller, 1997). Arabidopsis mutants that are either
comprised in their ability to biosynthesise JA or are
insensitive to JA are highly susceptible to herbivory and
certain pathogens (McConn et al., 1997; Vijayan et al.,
1998). Moreover, mutant plants are male sterile and
unable to complete their life cycle (Stintzi and Browse,
2000).
In 1962, methyl jasmonate (MeJA) was first identified
as an odorant compound in the essential oil of jasmine.
During the 80s, jasmonic acid (JA) was discovered as a
plant hormone-like substance influencing develop-
mental processes such as growth inhibition, induction of
senescence and tuber formation (Parthier, 1991). MeJA
and JA were found to occur apparently ubiquitously in
the plant kingdom (Hamberg and Gardner, 1992).
In 1990, Farmer and Ryan recognized the essential
role of JA as a signal molecule in plant defence (Farmer
and Ryan, 1990). When JA or MeJA are applied
* Corresponding author. Tel.: +49-931-888-6160; fax: +49-931-
888-6182.
E-mail address: martin.mueller@biozentrum.uni-wuerzburg.de
Wounding, pathogens and drought have all been
shown to transiently induce JA that can rapidly be
(M.J. Mueller).
0031-9422/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0031-9422(02)00126-7

234
C. Stuhlfelder et al. / Phytochemistry 60 (2002) 233–240
Here we report that MeJA cleaving esterase activity is
abundant in all plant species so far analysed. The MeJA
cleaving enzyme was purified, characterized and partial
protein sequence has been obtained by Edman sequencing.
2. Results and discussion
2.1. MeJA esterase activity in plant cell cultures
In total, 18 cell cultures were screened for MeJA
esterase activity. Typically, 5 g of plant tissue were
shock frozen in liquid nitrogen, grinded, suspended in
10 ml of buffer A and centrifuged (20 min, 12 000 g).
The supernatant was passed through a PD-10 desalting
column and MeJA esterase activity was determined in
the crude protein fraction using [methyl-3H]MeJA as
substrate. Release of [3H]MeOH was determined after
absorption of unreacted MeJA on a RP18-column by
scintillography. In some preparations, we also employed
methyl [2-14C]jasmonate as substrate to confirm the for-
mation of [2-14C]JA. Both, substrate and product could be
identified after TLC with a radioisotope TLC analyser.
MeJA esterase activity was detected in all plant tissues
so far analysed and ranged from 0.21–5.67 pkat/mg of
protein (Fig. 1). Cultured cells of Lycopersicon escu-
lentum were selected as a convenient plant source for
enzyme purification, since this cell culture displays
excellent growth characteristics yielding kilograms of
cell material each week.
Fig. 1. MeJA cleaving esterase activity in different plant cell cultures.
Esterase activity was determined from the soluble protein fraction
from different plant cell cultures after removal of low molecular weight
contaminants by PD-10 gel-filtration.
methylated to the volatile MeJA (Mueller, 1997). It has
been shown that movement of jasmonates among plants
may occur via the vapour phase in the form of MeJA
(Farmer and Ryan, 1990; Karban et al., 2000; Farmer,
2001). MeJA can readily be taken up by plant cells and
may be cleaved by an esterase to regenerate nonvolatile
JA within cells. Thus, a volatile long distance signal may
be generated by reversible methylation of JA. Alter-
natively, MeJA may represent itself a signal that needs
not to be hydrolyzed to JA to mediate defense gene
activation. Since the biological activities of MeJA and
JA can not be differentiated in vivo, these two possi-
bilities cannot be distinguished.
Wounding (Creelman et al., 1992) and pathogen
attack (Gundlach et al., 1992) often increases levels of
JA and MeJA almost simultaneously. Recently, a jas-
monic acid carboxyl methyltransferase has been cloned
and overexpressed in Arabidopsis thaliana (Seo et al.,
2001). Transgenic plants had 3-fold elevated levels of
endogenous MeJA without altered JA content. More-
over, mutant plants exhibited constitutive expression of
jasmonate-responsive genes and showed enhanced level
of resistance against the necrotrophic fungus Botrytis
cinera. Therefore, MeJA formed by jasmonic acid
methyltransferase may be an important signal com-
pound in addition to JA.
2.2. Purification of MeJA cleaving esterase
An outline of the purification of MeJA cleaving
esterase from L. esculentum is shown in Table 1. For
enzyme isolation and purification, 500 g of L. escu-
lentum cells were grown and harvested. The crude
extract obtained was subjected to (NH4)2SO4 fractiona-
tion followed by anion-exchange chromatography on
Q-Sepharose Fast Flow. At this stage 4.7-fold enzyme
enrichment with an overall yield of 48% was obtained.
After each chromatographic step, fractions containing
MeJA esterase activity were concentrated by ultrafiltra-
tion. A further 6-fold enrichment of MeJA cleaving
esterase with an overall yield of 30% was achieved when
the remaining protein from Q-Sepharose chromato-
graphy was subjected to a HiLoad Superdex 200 gel-
filtration column. This unusual step at this stage of
purification was necessary since a further reduction of
contaminating polyphenols could be efficiently accom-
plished through gel-filtration. Fractions with the highest
enzyme activity were combined and used for the next
purification step on a hydroxylapatite column. Elution
with a linear K-Pi gradient gave a 25-fold purification of
the MeJA esterase activity with a yield of 22.4%. Ion
exchange chromatography on a Bio-Scale Q2 column
Characterization of the enzyme catalysing the reverse
reaction, MeJA cleaving esterase, would help to clarify
the exact role and relative importance of MeJA and JA.

C. Stuhlfelder et al. / Phytochemistry 60 (2002) 233–240
235
Table 1
Purification of methyl jasmonate cleaving esterase
Purification step
Total protein
(mg)
Total activity
(pkat)
Specific activity
(pkat/mg)
Purification
(-fold)
Recovery
(%)
Crude extract
2503
794
4430
2985
2140
1341
992.7
804.3
89.2
1.77
3.76
1
2.167.4
4.7
100
(NH4)2SO4 (30–70%)
Q-Sepharose fast flow
HiLoad Superdex 200
Bio-Scale CHT5
Bio-Scale Q2
255
123
8.39
48.3
30.3
22.4
18.16
2.2
10.91
44.04
365.6
1357.1
6
25
22.5
2.2
207
767
Mono Q HR 5/5
0.0723
gave 207-fold enrichment with a specific activity of
365.6 pkat/mg. The remaining enzyme could be further
purified on a Mono Q column. During purification,
MeJA esterase activity was always found in one single
activity peak. In summary, the protein coeluting with
MeJA esterase activity was 767-fold purified and
showed a specific activity of 1.36 nkat/mg with a 2.2%
recovery from the crude protein extract.
revealed that esterase activity corresponded to the
stronger protein band with a Mr of ꢁ28 kDa on SDS-
PAGE. The accurate mass of this protein determined by
ESI-TOF MS was 28547.714 Da (s.d. 237.339 Da).
Since the smaller 25 kDa protein was of low abundance
we used this fraction for enzyme characterization.
Chromatofocussing of MeJA cleaving esterase on a
Mono P column gave an isoelectric point (pI) of 4.7 and
examination of the pH-dependence of enzyme activity
showed that the highest activity was at pH 9.0, whereas
50% of activity was at pH 5.5 and 10.0 (data not
shown). Highest activity of the esterase was observed at
a temperature of 40 ꢀC. MeJA cleaving esterase was
remarkably stable at room temperature and did not
loose enzyme activity even after storage under sterile
conditions at room temperature for several days.
No inhibition of the esterase was observed using the
cysteine protease inhibitors N-ethylmaleimide and
iodoacetamide at concentrations up to 10 mM. Addi-
tion of bestatin (65 mM) and pepstatin (1 mM), inhibi-
tors of metal and acid proteases, respectively, did not
inhibit MeJA cleaving esterase. In contrast, addition of
0.5 or 5 mM phenylmethylsulfonyl fluoride, a serine
protease inhibitor, yielded 50% and complete inhibition
of the esterase, respectively. MeJA cleaving esterase
obeys Michaelis–Menten kinetics from which apparent
Km (15.1Æ1.4 mM) and Vmax (7.97Æ0.5 nkat/mg) values
could be deduced. The Km value for MeJA stresses the
enzyme’s ability to work effectively with the expected low
intracellular concentrations of MeJA and is in the same
range as the Km (38 mM) for JA methyltransferase (Seo
et al., 2001). A turnover number of 0.23 sÀ1 was calcu-
lated for MeJA cleaving esterase. This value is far below
the turnover number found for JA methyltransferase
(25 sÀ1). Thus, the Kcat/Km value for the esterase (0.016
In order to obtain sufficient enzyme for partial
sequencing, enzyme purification was repeated from 3 kg
of L. esculentum cells.
2.3. Characterization of MeJA cleaving esterase
A molecular mass of 26 kDa for MeJA cleaving
esterase was determined by gel-filtration on a Sephacryl
S-100 column. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) analysis followed by
coomassie or silver staining showed that the fraction
purified through the Mono Q column gave two major
protein bands with a Mr of ꢁ28 and ꢁ25 kDa (Fig. 2).
Careful analysis of the fractions eluting before and after
the fraction with maximum MeJA esterase activity
mMÀ1
s
À1) is substantially lower than for the JA
À1) indicating that the
methyltransferase (0.7 mMÀ1
s
Fig. 2. SDS–PAGE analysis (12.5%, silver staining) of MeJA cleaving
esterase (MJE) preparations at different stages of purification. Lane 1:
crude extract; lane 2: (NH4)2SO4 fraction (30–70% saturation); lane 3:
Q-Sepharose fast flow; lane 4: HiLoad Superdex 200; lane 5: Bio-Scale
CHT5; lane 6: Bio-Scale Q2; lane 7: Mono Q HR 5/5; lane 8: mole-
cular weight markers. The arrow indicates the protein band corre-
sponding to MeJA cleaving esterase.
methyltransferase works more efficient than the esterase
at low jasmonate concentrations.
Substrate specificity of MeJA cleaving esterase was
tested with various ester compounds at a concentration
of 0.5 mM under the standard enzyme reaction con-
ditions described in the Experimental section. MeJA

236
C. Stuhlfelder et al. / Phytochemistry 60 (2002) 233–240
cleaving esterase efficiently hydrolyzed in addition to
MeJA, methyl esters of abscisic acid, indole-3-acetic
acid, linolenic acid and linoleic acid as well as the
chromogenic substrate p-nitrophenyl acetate (Table 2).
In contrast, methyl esters of salicylic acid, benzoic acid
and cinnamic acid were poor substrates. The amide,
leucine-p-nitroanilide, was not hydrolyzed.
2.4. Partial amino acid sequence of the esterase
The proteins with an apparent Mr of 25 and 28 kDa
obtained from SDS-PAGE were digested with protei-
nase LysC and the resulting peptides were separated by
HPLC and sequenced. Peptides of the 25 kDa protein
were identified as fragments of an abundant tomato
heat shock protein, HSP70, by database search. The
putative esterase (28 kDa) yielded three peptide frag-
ments (PF) from which their peptide sequences could be
obtained (Table 3). Sequencing of the N-terminus of the
ꢁ28 kDa protein band gave a fourth peptide fragment
which lacked the first N-terminal amino acids, probably
due to partial proteolytic degradation. Comparison of
the four peptide sequences against protein databases
(NCBI and SWISS-PROT) revealed that the esterase is
not yet annotated in the databases. However, all four
fragments displayed 36–82% sequence identity to dif-
ferent plant esterases such as polyneuridine aldehyde
esterase from Rauvolfia serpentina, ethylene-induced
esterase from Citrus sinensis and the pathogen-inducible
protein Pir 7b from Oryza sativa as well as cyanohydrin
lyases (Table 4). Interestingly, all of these proteins
belong to the a/b-hydrolase super family, which is
characterized by a conserved arrangement of the com-
ponents of the catalytic triad. The catalytic triad—
nucleophile (Ser, Cys or Asp), acid (Asp or Glu) and a
histidine—form the basis of the enzymatic properties of
this class of enzymes. Despite high structural homology
among different members of this protein family, a low
degree of sequence conservation is usually found.
Enzymes of this family also display different enzymatic
activities. Besides esterases and hydroxynitrile lyases,
haloalkane dehalogenases, semialdehyde hydrolases,
Table 2
Relative hydrolase activity of tomato MeJA cleaving esterase on various
esters and amides
Substrates
Relative activity
(meanÆS.D.) (%)
(Æ)Jasmonic acid methyl ester (MeJA)
Indole-3-acetic acid methyl ester
Linolenic acid methyl ester
Acetic acid p-nitrophenyl ester
(Æ)Abscisic acid methyl ester
Linoleic acid methyl ester
100.0Æ1.0
95.9Æ1.7
71.7Æ12.6
65.3Æ11.6
32.4Æ6.6
12.4Æ2.7
4.5Æ0.6
3.3Æ2.7
2.9Æ0.8
0
Cinnamic acid methyl ester
Benzoic acid methyl ester
Salicylic acid methyl ester
L-Leucine p-nitroanilide
All substrates were tested at a 0.5 mM concentration. Incubations were
terminated by addition of hydrochloric acid. Substrates and products were
extracted with diethyl ether and treated with diazoethane. Aliquots were
analyzed by GC-positive chemical ionization-MS and methyl esterase
activity was calculated from the peak areas of the [M+H+] ions of the
corresponding methyl esters (unreacted substrate) and ethyl esters (product
derivative). In the case of the chromogenic substrates (p-nitrophenyl ester
and p-nitroanilid) product formation was determined photometrically. The
relative esterase activity with MeJA was set arbitrarily at 100%. Values are
the meanÆS.D. from three independent experiments.
Table 3
Sequences of four peptide fragments (PF) of MeJA cleaving esterase
Peptide fragment Amino acid sequence
PF 1(N-terminus) Lys-Gly-Asp-Lys-Asn-His-Phe-Val-Leu-(X)-His
PF 18 a
PF 18 b
PF 23
Val-Val-Thr-Ile-Leu-Arg-Ser-Glu-Gly-His-Lys
Tyr-Gly-Ser-Val-His-Arg-Val-Tyr-Val-Val-Cys-Asp-Lys
Val-Ser-Val-Leu-Asp-Met-Ala-Ala-Ser-Gly-Ile-Asn-Pro-Lys
Table 4
Sequence comparison of MeJA cleaving esterase peptide fragments against SWISS-PROT database using FASTA3
Enzymes
Sequences with homologies to N-terminus
Identity (%)
Sequences with homologies to PF 18a
Identity (%)
KGDK–NHFVLxH
VVTILRSEGHK
RS
CS
ME
OS
1MHSAANA KQQKHFVLVH
1MEEVVGMEEK HFVLVH
1VTA— HFVLIH
60
50
50
40
29 LKPLLESAGHK 39
28 LKARLVAGGHR 38
21LKPALERAGHK 31
27 VVTMLRSEGHR 37
46
27
36
1megsssssk hfilvh
82
Enzymes
Sequences with homologies to PF 18b
Identity (%)
Sequences with homologies to PF 23
Identity (%)
VSVLDMAASGINPK
40 VTAVDLSAAGINPR53
39 VTAVDLAASGINMK52
YGSVHRVYVVCDK
RS
CS
ME
OS
43
64
203YGSVKRAYIFCNE215
204YGSVKRVYLVCEE216
YGSIKKVYIWTDQ206
YGSVKRVCLMAME166
54
69
46
46
32 VTALDMAASGIDPR45
38 VTALDLAASGVHPA515574
94
711
1
Enzymes are: RS, polyneuridine aldehyde esterase from Rauvolfia serpentina (Q9SE93); CS ethylene-induced esterase from Citrus sinensis (Q94G63); ME, (S)-
Acetone-cyanohydrin lyase from Manihot esculenta (P52705); OS, Pir 7b (Pseudomonas inducible protein) from Oryza sativa (Q942Y9). SWISS-PROT accession
numbers are given in brackets; numbers indicate location of peptides in the enzyme. Identical amino acids are bold.

C. Stuhlfelder et al. / Phytochemistry 60 (2002) 233–240
237
haloalkane peroxidases and epoxide hydrolases have
been found within this structural class (Heikinheimo et
al., 1999).
The sequence information reported herein can be used
to clone the corresponding esterase gene from tomato.
In future, transgenic plants may be produced that are
either devoid of MeJA esterase activity or overexpress
the MeJA cleaving esterase in order to study the func-
tion of the enzyme in plant defence signalling.
of Et2O. The flow through and the eluate containing
[methyl-3H]MeJA were collected and taken to almost
dryness under a gentle stream of nitrogen. Remaining
solvent was allowed to evaporate at room temperature
without any manipulations, since considerable losses of
labelled MeJA may occur during intense drying. This
special precautions are necessary since the boiling point
ꢀ
of MeJA is only 110 C.
Labelled MeJa was analysed by thin layer chromato-
graphy (hexane–Et2O–HOAc, 70:30:1) using unlabelled
MeJA and JA as references. The absence of unreacted
JA was confirmed by staining with iodine vapour.
Radiometric analysis revealed that more than 98% of
the radioactivity comigrated with methyl jasmonate.
The synthesis yielded 19.9% racemic [methyl-3H]MeJA.
Racemic [2-14C]JA was synthesized from from 2-(2Z-
pentenyl)-2-cyclopenten-1-one and diethyl [2-14C]mal-
onate followed by saponification and decarboxylation
of the Michael product in one step as described (Knofel
and Gross, 1988). [2-14C]MeJA was prepared from label-
led JA by incubation with diazomethane in diethyl ether.
3. Experimental
3.1. General experimental procedures
JA methyl ester (racemic mixture) was obtained from
Serva (Heidelberg, Germany). [Methyl-3H]methyl
iodide (sp. act of 85 Ci/mmol) was from Amersham
Pharmacia Biotech (Freiburg, Germany). Solid phase
extraction columns (1ml) were from Amchro (Hatter-
sheim, Germany). Aminopropyl- and RP18-solid phase
extraction materials and Polygram SIL G/UV254 0.2
mm thin layer chromatography plates were from
Macherey-Nagel (Duren, Germany). Scintillation cock-
tail Ultima Gold was purchased from Packard (Gro-
ningen, The Netherlands). For ultrafiltration, Centricon
Plus-20 centrifugal filter devices (membrane type PL-10)
from Millipore (Eschborn, Germany) were used.
Q-Sepharose Fast Flow material, Superdex 200 prep
grade material, Mono Q HR 5/5 column, Mono P HR
5/20 column, Sephacryl S-100 HR 26/60 column and
PD-10 desalting columns were from Amersham Phar-
macia Biotech (Freiburg, Germany). Bio-Scale CHT5
column and Bio-Scale Q2 column were from Bio-Rad
Laboratories (Munich, Germany).
3.4. Enzyme assays
Standard enzyme incubation mixtures (total volume
30 ml) contained enzyme, 167 mM of [methyl-3H]MeJA
(sp. act of 9.09 mCi/mmol) in 50 mM K-Pi buffer at pH
ꢀ
7.5. Incubations were performed at 37 C for the times
indicated (5–60 min). Reactions were terminated by
addition of 20 ml of HOAc and 100 ml of H2O–MeOH
(70:30). The mixture was loaded on a RP18-solid phase
extraction column (50 mg) equilibrated with H2O–
MeOH (90:10). [3H]MeOH released from the enzyme
was eluted with 900 ml of H2O–MeOH (90:10) and
radioactivity was quantified by scintillation counting
after addition of 2 ml of scintillation cocktail. Internal
standards were used to correct for quenching by MeOH.
Substrate specificity was measured by incubating the
purified enzyme with various unlabelled methyl esters at
a concentration of 500 mM in a total volumne of 50 ml
for 30 min. After an incubation time of 30 min, 10 ml of
1M hydrochloric acid and 500 ml of diethyl ether were
added. The mixture was mixed and centrifuged. The
organic layer containing the substrate (methyl ester) and
product (free acid) was separated and treated with an
etheral solution of diazoethane. The solution was eva-
porated to 50 ml and one ml was analyzed by GC–MS in
the positive chemical ionization mode as described
(Gundlach et al., 1992). Product formation was calcu-
lated from the peak areas of the [M+H]+ ions of the
methyl ester (substrate) and the ethyl ester (representing
the derivative of the free acid). In the case of chromo-
genic substrates, product formation was measured
photometrically. The relative activity of MeJA cleaving
esterase with MeJA (50% substrate consumption in 30
min) was set arbitrarily to 100%.
The other chemicals used were of highest purity.
3.2. Plant cell material
Cell suspensions cultures of L. esculentum were grown
in 1l Erlenmeyer flasks in Linsmaier and Skoog medium
(Linsmaier and Skoog, 1965) for 7 days under continuous
light (600 lux) on orbital shakers (100 rpm) at 24Æ2 ꢀC.
The cells were harvested by suction filtration, shock frozen
with liquid nitrogen, and stored atÀ20 ꢀC until use.
3.3. Synthesis of [methyl-3H]MeJA and of [2-14C]JA
Racemic jasmonic acid was prepared by alkaline
hydrolysis of the methyl ester (Gundlach et al., 1992).
For synthesis of [methyl-3H]MeJA, 500 mg of JA was
dissolved in 100 ml of EtOAc and incubated with 1mCi
[methyl-3H]methyl iodide (sp. act of 8.5 Ci/mmol) and 1
mg of K2CO3 in a tightly locked 0.5 ml vial at 30 ꢀC for 90
min. The mixture was loaded on an aminopropyl-solid
phase extraction column (200 mg) and eluted with 1ml

238
C. Stuhlfelder et al. / Phytochemistry 60 (2002) 233–240
In some assays, JA formation from MeJA was con-
3.9. Fractional precipitation with (NH4)2SO4
firmed by using Me[2-14C]JA as substrate. Formation of
[2-14C]JA by the esterase was detected with a radio-
isotope TLC analyser. The standard assay solution
(total volume 200 ml) consisted of 50 mM K-Pi buffer,
pH 7.5, and 50 mM [2-14C]MeJA (sp. act of 0.77 mCi/
mmol). After an incubation time of 30 min at 37 C, the
reaction was terminated by adding 20 ml HOAc and 400
ml EtOAc. The mixture was mixed and centrifuged. The
organic phase was separated and carefully concentrated
under a gentle stream of nitrogen. Since MeJA evapo-
rates after prolonged time under a nitrogen stream, the
organic phase was not completely evaporated. The con-
centrate was analysed by a radioisotope TLC analyser
on silica plates using hexane–EtOAc–HOAc (70:30:1) as
solvent.
The protein in the resulting supernatant was subjected
to (NH4)2SO4 precipitation. The protein fraction pre-
cipitating between 30–70% saturation was collected
after centrifugation (20 min, 12 000 g), redissolved in a
minimal volume of buffer B and desalted by passage
through PD-10 desalting columns equilibrated pre-
viously with buffer B yielding the soluble protein frac-
tion (90 ml, 8.8 mg/ml).
3.10. Anion-exchange chromatography on Q-Sepharose
fast flow
The soluble protein fraction was loaded on a
Q-Sepharose fast flow anion-exchange column (2.6Â6
cm) previously equilibrated with buffer B. The column
was washed with 250 ml of buffer B and MeJA cleaving
esterase activity was eluted with a 50 ml linear KCl-
gradient prepared from buffers B and C at a flow rate of
4 ml/min. Fractions containing MeJA cleaving esterase
activity (>3 pkat/mg) were combined (24 ml), desalted
and concentrated to 3 ml by ultrafiltration.
3.5. Protein determination
Protein concentration was determined by the method
of Bradford using bovine serum albumin (Bradford,
1976).
3.6. Composition of buffers
3.11. Gel-filtration on HiLoad 16/60 Superdex 200 prep
grade
Buffer A: 50 mM K-Pi (pH 7.5), 20 mM b-mercap-
toethanol (EtSH), 10 mM ascorbic acid and 1.5%
PVPP; Buffer B: 50 mM K-Pi (pH 7.5), 20 mM EtSH;
Buffer C: buffer B containing 1M KCl; Buffer D: buffer
B containing 0.15 M NaCl; Buffer E: 20 mM K-Pi (pH
6.8), 20 mM EtSH, 0.3 mM CaCl2; Buffer F: 500 mM
K-Pi (pH 6.8), 20 mM EtSH. Buffer G: 50 mM tricine
buffer (pH 8), 20 mM EtSH; Buffer H: buffer G con-
taining 1M KCl; Buffer I: 50 mM K-Pi (pH 5.5), 20
mM EtSH; Buffer J: buffer I containing 1M KCl; Buf-
fer K: 25 mM bis–Tris set to pH 6.3 with HCl; Buffer L:
10% polybuffer 74 (Amersham Pharmacia Biotech,
Freiburg) at pH 4.0 adjusted with HCl.
For gel-filtration, the above enzyme solution was
chromatographed on a 1.6Â60 cm column containing
Superdex 200 prep grade material equilibrated with
buffer D. The MeJA cleaving esterase was eluted with
buffer D at a flow rate of 1ml/min and fractions con-
taining esterase activity (>4 pkat/mg) were combined
(35 ml). Sample concentration (1ml) and buffer
exchange to buffer E were accomplished by ultrafiltration.
3.12. Bio-Scale CHT5 ceramic hydroxyapatite
chromatography
3.7. Purification of MeJA cleaving esterase
The Bio-Scale CHT5 column (1.0Â6.4 cm) was equi-
librated with buffer E. Active protein fractions from gel-
filtration columns were applied to a Bio-Scale CHT5 col-
umn with a flow rate of 1ml/min. After washing the col-
umn with buffer E for 12 min, proteins were eluted with a
gradient (25 min) prepared from buffer E and F. Frac-
tions exhibiting MeJA cleaving esterase activity (>10 pkat/
mg) were combined (7 ml) and concentrated to 1ml by
ultrafiltration. Sample buffer was exchanged to buffer G.
All operations were performed at 4 ꢀC. Desalting,
concentration of the enzyme solution and buffer
exchange between all chromatographic steps was per-
formed by ultrafiltration using Centricon Plus-20 centri-
fugal filter devices.
3.8. Preparation of crude protein extracts
Cells of L. esculentum (500 g, fr. wt) were shock fro-
zen with liquid nitrogen, added to 1l of buffer A and
homogenised with a Waring blender operated at max-
imum speed for 5 min. The homogenate was filtered
through cheesecloth and clarified by centrifugation (20
min, 12 000 g). The supernatant (1500 ml of crude extract)
was used immediately for the next purification step.
3.13. Bio-Scale Q2 anion-exchange chromatography
The above obtained protein solution was loaded on a
Bio-Scale Q2 anion-exchange column (0.7Â5.2 cm)
equilibrated with buffer G. After washing the column
for 8 min at a flow rate of 1ml/min, proteins were
eluted with a linear KCl gradient (0–1.0 M) prepared

C. Stuhlfelder et al. / Phytochemistry 60 (2002) 233–240
239
from buffer G and H. Fractions (>90 pkat/mg) were
combined (7 ml) and concentrated to 1ml using ultra-
filtration. Sample buffer was exchanged to buffer I.
To determine the optimal temperature for hydrolysis,
standard esterase assays using MeJA as subꢀstrat were
performed at temperatures between 5 and 70 C.
Kinetic data were fitted to standard equations using
the software package Origin. Vmax and apparent Km
values were determined from the Lineweaver–Burk plot.
For inhibition studies MeJA cleaving esterase (100
mg/ml) was preincubated with different concentrations
of inhibitor at room temperature for 15 min. Phenyl-
methylsulfonyl fluoride, N-ethylmaleimide, iodoaceta-
mide, pepstatin were dissolved in EtOH and bestatin
was dissolved in MeOH. All inhibitors were diluted with
50 mM K-Pi buffer (pH 6.5, EtSH-free) to the con-
centration needed. Esterase activity assay was performed
as described under Section 3.4 for 15 min at 37 ꢀC. Resi-
dual MeJA cleaving esterase activity was expressed as
percentage of a control preincubation without inhibitor.
3.14. Mono Q HR 5/5 anion-exchange chromatography
The remaining protein solution from the anion-
exchange chromatography was loaded on a Mono Q
column (0.5Â5 cm) with a flow rate of 1ml/min. The
column was washed with buffer I for 8 min. The enzyme
was eluted with a linear KCl-gradient (0–1.0 M) pre-
pared from buffer I and J at a flow rate of 1ml/min.
Fractions of 0.5 ml were collected. Enzyme activity was
found in fractions containing 200–300 mM KCl and
active fractions (>500 pkat/mg) were combined.
3.15. FPLC-chromatofocussing with Mono P HR 5/20
column
3.18. Sequencing of MeJA cleaving esterase and peptide
sequence alignment
For determination of the isoelectric point (pI), the
protein solution was loaded on a Mono P HR 5/20 col-
umn (0.5Â20 cm). Proteins were fractionated (1.0 ml)
with 50 ml buffer L at a flow rate of 0.7 ml/min gen-
erating a linear pH gradient from pH 6.3 to 4.0. The
enzyme eluted after 30 ml of buffer L at a pH of 4.7.
MeJA cleaving esterase enriched protein fraction
obtained from Mono Q chromatography was applied to
SDS–PAGE (12.5%) and Coomassie stained. The
dominating protein band at 28 kDa and a minor protein
band at 25 kDa were cut out and destained with 10%
HOAc. Gel slices were digested with endoproteinase
LysC. The resulting peptides were separated by HPLC
on a Supersphere 60 RP select B column (Merck,
Darmstadt) and sequenced.
For N-terminal sequencing, the 28 kDa protein band
was blotted on a PVDF membrane by the semi dry
blotting method and sequenced.
Peptide sequences were used to screen the SWISS-
PROT sequence library using the sequence analysis
program FASTA3.
3.16. Molecular weight determinations by Sephacryl S-
100 chromatography, sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) and
electrospray ionization time of flight mass spectrometry
(ESI-TOF MS)
The native molecular mass of the enzyme was estimated
by gel-filtration with a Sephacryl S-100 HR 26/60 column
(2.6Â60 cm) equilibrated with buffer D. The column was
calibrated with transferrin (80 kDa), bovine serum albu-
min (67 kDa), ovalbumin (43 kDa), chymotrypsinogen
(25 kDa) and ribonuclease a (13.7 kDa) as standard
proteins (Amersham Pharmacia Biotech, Freiburg).
Molecular weight under denaturating conditions was
determined by SDS–PAGE (12.5%) analysis.
ESI-TOF MS analyses were performed on an ETTAN
LC–MS system from Amersham Pharmacia Biotech
(Freiburg, Germany). The protein sample was desalted
and concentrated on a Trap-Column from Amersham
Pharmacia Biotech (Freiburg, Germany) prior ESI-
TOF MS analysis. M+-Spectra were calculated using
the software package MagTrans.
Acknowledgements
We thank Reinhard Mentele (Max-Planck-Institute
for Biochemistry, Martinsried) for performing peptide
sequence analysis. We also thank Axel Parbel, Amer-
sham Pharmacia Biotech, Freiburg, for ESI-TOF–MS
analysis of the protein. We are also indepted to Martina
Ruffer (Pharmaceutical Biology, Munich) for valuable
advice and support.
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