Brassica napus soluble epoxide hydrolase BNSEH1Cloning and characterization of the recombinant enzyme expressed in Pichia pastoris Stefan Bellevik1, Jiaming Zhang2and Johan Meijer1 1 Dep
Trang 1Brassica napus soluble epoxide hydrolase (BNSEH1)
Cloning and characterization of the recombinant enzyme expressed in
Pichia pastoris
Stefan Bellevik1, Jiaming Zhang2and Johan Meijer1
1 Department of Plant Biology, Genetics Center, Swedish University of Agricultural Sciences, Uppsala, Sweden; 2 National
Biotechnology Laboratory of Tropical Crops, Chinese, Academy of Tropical Agricultural Sciences, Chengxi, Haikou, China
Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the
metabolism of epoxy fatty acids and in mediating defence
responses We report the cloning of a full-length epoxide
hydrolase cDNA (BNSEH1) from oilseed rape (Brassica
napus) obtained by screening of a cDNA library prepared
from methyl jasmonate induced leaf tissue, and the 5¢-RACE
technique The cDNA encodes a soluble protein containing
318 amino acid residues The identity on the protein level is
85% to an Arabidopsis soluble epoxide hydrolase (sEH) and
50–60% to sEHs cloned from other plants A 5· His tag
was added to the N-terminus of the BNSEH1 and the
con-struct was over-expressed in the yeast Pichia pastoris The
recombinant protein was recovered at high levels after
Ni-agarose chromatography of lysed cell extracts, had a
molecular mass of 37 kDa on SDS/PAGE and cross-reacted
on Western blots with antibodies raised to a sEH from Arabidopsis thaliana BNSEH1 was shown to be a monomer
by gel filtration analysis The activity was low towards cis-stilbene oxide but much higher using trans-cis-stilbene oxide as substrate with Vmaxof 0.47 lmolÆminÆmg)1, Kmof 11 lM
and kcat of 0.3 s)1 The optimum temperature of the recombinant enzyme was 55C and the optimum pH 6–7 for trans-stilbene oxide hydrolysis The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop
Keywords: epoxide hydrolase, oilseed rape, yeast, recom-binant enzyme, stilbene oxide
Epoxide hydrolases [1] are hydrolytic enzymes that have
been found in mammals, plants, yeast, fungi, insects and
bacteria In mammals several forms exist with a
heteroge-neous tissue distribution of which microsomal and soluble
(sEH) epoxide hydrolases have been most extensively
studied These enzymes differ from each other in several
aspects such as substrate preference, turnover rate,
sensitiv-ity to inhibitors, pH optimum, etc [2] Several geometrically
different but related epoxides such as cis-stilbene oxide
(CSO) and trans-stilbene oxide (TSO) have been found to be
useful substrates in order to distinguish soluble from
membrane bound epoxide hydrolases [3,4] These substrate
pairs can be applied to crude extracts to assess the relative
contribution of membrane bound and soluble forms to the
total epoxide hydrolase activity in many species
Based on sequence homology analysis epoxide hydrolase
was classified as a member of a super family of hydrolytic
enzymes including esterases and lipases, united by an a/b
hydrolase fold and a similar catalytic triad motif [5] The
three-dimensional structure of epoxide hydrolase has been resolved for mouse [6], fungal [7] and bacterial enzymes [8] These studies have confirmed the predicted a/b fold struc-ture, provided a detailed picture of the active site and proposed a mechanism of the catalytic reaction supported by site-directed mutagenesis Epoxide hydrolases act through a two-step mechanism in which an acidic nucleophile attacks the epoxide ring, forming a covalent intermediate, which is then hydrolysed by a polarized water molecule [9]
Epoxide hydrolases in mammals are essential in the detoxification of epoxides that are toxic due to the electrophilic and unstable nature of the epoxide ring [2] The relevance of this enzyme for detoxification in plants is uncertain, however Certain plants store epoxy fatty acids in seeds, e.g Euphorbia lagascae contains up to 60% epoxy fatty acids in the seed oil [10] Epoxide hydrolase is probably needed for complete b-oxidation of epoxy fatty acids, e.g during germination when seed stores are broken down prior
to photosynthetic growth However, epoxide hydrolases are also present in plants low in epoxy fatty acids and have been cloned from several species such as potato [11], soybean [12,13], Arabidopsis [14] and Euphorbia [15] No microsomal epoxide hydrolase has been cloned from plants as yet, which indicates that this form is not necessary or that sEH may have a broader role in general
Engineering of epoxide hydrolase in a crop such as oilseed rape allows for agricultural and industrial applications Polymers of epoxy and hydroxy fatty acids are important constituents of the cutin layer and serve as a protective barrier against stress [16] Certain epoxy and hydroxy fatty acids have fungicidal activity in rice [17] and mediate defence responses in infected potato tubers [18] Modulation of
Correspondence to S Bellevik, Department of Plant Biology,
Genetics Center, Box 7080, Swedish University of Agricultural
ScI`ences, SE-750 07 Uppsala, Sweden.
Fax: + 46 18 673279, Tel.: + 46 18 673320,
E-mail: Stefan.Bellevik@vbiol.slu.se
Abbreviations: BNSEH1, soluble epoxide hydrolase 1 from Brassica
napus; CSO, cis-stilbene oxide; MeJa, methyl jasmonate; sEH, soluble
epoxide hydrolase; TSO, trans-stilbene oxide.
Enzymes: epoxide hydrolase (EC 3.3.2.3).
(Received 26 June 2002, revised 27 August 2002,
accepted 10 September 2002)
Trang 2epoxide hydrolase expression may thus be used to improve
crop protection Another possibility is the use of B napus as
a host to generate high amounts of epoxy or hydroxy fatty
acids in the seed for production of technical oils or plastics
[19] Successful engineering has already altered seed fatty acid
composition to create high laurate [20] and up-regulated
palmitate, stearate and c-linolenate (x-6) B napus lines [21]
Several genes encoding sEH exist in potato, soybean and
Arabidopsis[11,13,22] and apparently also in oilseed rape
(S Bellevik, J Lin & J Meier) Enzymatic characterization
is necessary to better understand the functional roles of the
isoforms and in this study we have used the yeast P pastoris
as host for over-expression of the first sEH cloned from
B napus(BNSEH1) We here present data concerning the
physico-chemical and biochemical properties of the
recom-binant enzyme
E X P E R I M E N T A L P R O C E D U R E S
Materials
Oligonucleotides were purchased from TAGC
(Copenha-gen, Denmark) All enzymes were purchased from MBI
Fermentas (Vilnius, Lithuania) unless stated otherwise
CSO and TSO (Aldrich Chemical Co., Milwaukee, WI,
USA); b-naphtoflavone, sodium-parahydroxymercuri
benzoate, quercitin (3,3¢,4¢,5,7-pentahydroxyflanone) (Sigma
Chemical Co., St Louis, MO); chalcone oxide (Lancaster
Synthesis, Morecambe, UK);
x-bromo-4-nitroacetophe-none (Fluka AG; Buchs, Switzerland);
1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (Bio-Rad Laboratories, Hemel
Hempstead, UK); N,N¢-dicyclohexylcarbodiimide (kind gift
of A˚ke Engstro¨m, Uppsala University); dimethylsulfoxide
(Merck AG, Darmstadt, Germany); tetrahydrofurane
(Riedel-de-Ha¨en, Seelze, Germany) were purchased from
the sources indicated N,N¢-dicyclohexylcarbodiimide was
hydrolysed at 37C for 16 h to form the corresponding
urea (N,N¢-dicyclohexylurea) derivative [23]
Cloning ofB napus sEH
One partial B napus sEH cDNA clone (BNSEH1) was
isolated by hybridization screening of a reamplified
(oli-godT)-primed cDNA library prepared from rapeseed leaves
exposed to methyl jasmonate (MeJa) for 5 h (Clontech,
Palo Alto, CA, USA) using a heterologous EST clone
(#140O6t7, Arabidopsis Ohio Stock Center) as a probe This
EST corresponds to an earlier described Arabidopsis
epo-xide hydrolase cDNA (AtsEH1) [14] but also contains
intron sequences (S Bellevik & J Meier) BB4 was used as
bacterial host strain in the hybridization screening and
XL1-Blue for conversion to pXL1-Bluescript The probe was
random-primed using [32P]dCTP (Amersham International, Bucks,
England), purified by gel filtration and used at 106
c.p.m.ÆmL)1 Hybridization was performed at high
strin-gency conditions using Hybond N+nylon filters
(Amer-sham Biosciences, Uppsala, Sweden) according to the
manufacturer’s instructions Signals were detected after
36–56 h exposure on Biomax MS X-ray films (Amersham
Biosciences) One of the positive phage clones was excised
from Lambda Zap II using R408 helper phage coinfection
but was found to lack the initial bases of the 5¢-end
(including the initiating methionine codon)
5¢-Rapid amplification of cDNA ends (RACE)
A SMARTTMRACE cDNA Amplification kit (Clontech) was employed to amplify the missing 5¢-region of the cDNA Total RNA from young B napus cv Hanna seedlings were isolated after grinding in liquid nitrogen using phenol/chloroform extraction and precipitation with lithium chloride Several RACE PCR products correspond-ing to the cDNA clone were obtained uscorrespond-ing one nested primer pair after the first cDNA synthesis In the first strand synthesis Superscript IITMRnase H–Reverse Transcriptase (Life Technologies, Ta¨by, Sweden) was used with the RACE kit The first PCR was performed with the Advant-age 2 Polymerase Mix supplied using the Universal Primer Mix and a gene specific primer (5¢ AGG ACC GAA AGA GAA AGG AAC AGA 3¢) by cycling for 35 cycles at 94 C for 10 s, 65C for 20 s and 72 C for 1 min The first PCR reaction was diluted 50-fold and 5 lL used in the second PCR The primers used for the nested reaction used the nested universal primer and a second gene specific primer (5¢ TGC GAA AAG ACA AAG ATA CCA AGC G 3¢) with a thermo profile as in the first PCR but for 30 cycles
No product could be visualized in the first PCR but in the nested reaction a 400-bp band appeared on an ethidium bromide stained agarose gel The 5¢-RACE products were sequenced on an ABI PrismTM 377XL DNA sequencer based on a D YEnamicTMET kit (Amersham Biosciences) The cDNA sequence has been deposited in the EMBL database under the accession number AJ459780
Cloning of BNSEH1 into pPIC3.5K and expression
inP pastoris For over-expression we used the P pastoris expression system (Invitrogen, Groningen, the Netherlands) The complete BNSEH1 cDNA was generated with a forward primer (5¢ AGA ATG GGA TCC ACC ATG GAT CAC CAT CAC CAT CAC ATG GAG CAC CGA AAG TTA AGA GGT AAC GG 3¢) containing five His codons, a BamH1 site, a Kozak consensus sequence for a proper translation initiation in P pastoris and also the nine initial bases missing in the isolated cDNA clone The reverse primer (5¢ AAG GTA GGA ATT CCT AGA ATT TGG AGA TGA AGT C 3¢) contained an EcoR1 site after the stop codon The PCR product was amplified using Taq DNA polymerase (Stratagene, La Jolla, CA, USA) and blunt-end cloned into the pPCR-Script AMP SK(+) cloning vector After sequencing the chosen clone was digested with BamH1 and EcoR1, transformed into E coli
by electroporation and subcloned into the P pastoris expression vector pPIC3.5K (Invitrogen) JM106 electro-competent cells were transformed, the selected clone cultured and prepared by a Qiagen plasmid Midi kit, linearized with Stu1 and purified with a PCR purification kit (Qiagen, Hilden, Germany) Competent GS115 yeast cells were electroporated with 15 lg of linearized plasmid before plating onto selective media The resulting colonies were tested in liquid culture for epoxide hydrolase activity after
24 h of methanol induction (calculated as percentage substrate turnover/ODcell suspension) using the [3H]TSO assay [3] Selected clones were grown in 2-L Fernbach flasks under vigorous agitation to improve aeration and cells were harvested after 4 days of methanol induction, centrifuged at
Trang 33000 g for 5 min and stored at)80 C if not processed at
once
Purification of recombinant BNSEH1
The frozen cell pellet was thawed and dissolved in start
buffer (20 mMTris/HCl, 40 mMimidazole, 500 mMNaCl,
1 mMbenzamidine, 1 mMphenylmethanesulfonyl fluoride,
20 mM2-mercaptoethanol, pH 7) and cells ruptured by at
least three passages through a 40K French pressure cell
press (SLM Aminco, Urbana, IL) prior to centrifugation
at 12 000 g for 10 min at 4C The supernatant was
sterile filtrated using 1.2 lm and 0.45 lm filters
HiTrapTM Chelating columns (Amersham Biosciences)
were loaded with 0.1M NiCl prior to addition of sample
The column was washed with start buffer and the enzyme
eluted with 150 mM imidazole The optimal imidazole
concentrations needed to remove contaminating proteins
in the washing step and to elute the recombinant enzyme
in the elution step were determined in preliminary
experiments Fractions were analysed for epoxide
hydro-lase activity and protein content based on the Peterson
procedure [24] using BSA as a standard, and also by SDS/
PAGE [25]
Western Blot analysis
Proteins were separated by SDS/PAGE, using 12.5%
polyacrylamide gels (Invitrogen) After completion of the
gel electrophoresis, separated proteins were detected by
Coomassie Brilliant Blue or by immunoblotting after wet
transfer to poly(vinylidene difluoride) filters (Schleicher &
Schull, Dassel, Germany) After blocking in milk/BSA,
filters were incubated with affinity purified rabbit polyclonal
anti-sEH Ig followed by HRP-conjugated swine anti-rabbit
Ig (Dako A/S, Glostrup, Denmark) and bands detected by
diaminobenzidine staining or ECL (Pierce, Rockford, IL,
USA) Antibodies were raised by immunization of rabbits
with recombinant sEH from A thaliana (AtsEH1) The
AtsEH1 corresponds to the EST clone used as probe for the
library screening (see above) Preimmune serum served as
the negative control The regional ethical committee
approved the animal experiments
Oligomerization analysis of BNSEH1 by gel filtration
A HiPrep 26/60 Sephacryl S-100 High Resolution column
(Amersham Biosciences) was chosen for native gel filtration
analysis of BNSEH1 dissolved in 0.1M potassium
phos-phate pH 7.2, 1 mM dithiothreitol The standard proteins
chymotrypsin A (horse heart, 1.8 mg), Cytochrome c
(1.8 mg), BSA (3.6 mg) and catalase (2.8 mg) (all from
Amersham Biosciences) were applied at a flow rate of
0.8 mLÆmin)1 The BNSEH1 (60 lg) was loaded at a total
volume of 500 lL, fractions collected and assayed for
enzyme activity and absorbance at 280 nm
Catalytic characterization of recombinant BNSEH1
Epoxide hydrolase activity was assayed based on conversion
of [3H]TSO or [3H]CSO synthesized as described [3] The
routine assays contained 100 lMfinal substrate
concentra-tion in 0.1 potassium phosphate, pH 7.0 and were run at
30C during a time period that assured significant activity within the linear range All measurements were performed
in at least duplicates and repeated twice or more The pH dependence of the enzyme for TSO hydrolysis was tested in potassium phosphate buffers (pH 4.5–7), Tris/HCl buffers (pH 7–9) and glycine/NaOH buffers (pH 9–11) pKavalues were obtained from intersections of asymptotes to the curve plot of log(kcat) vs pH Activation enthalpy was calculated from Arrhenius plots of ln(kcat) vs 1/T based on values up
to the denaturation point (55C) For the kinetic param-eters a range of approximately 10-fold higher and lower substrate concentrations of the predicted Kmwas tested and substrate conversions were kept below 5% to obtain accurate estimates of initial velocities The data were used
to draw a Lineweaver-Burk plot and the line equation was used to calculate Kmand Vmaxvalues When inhibitors were tested the inhibitor was preincubated with enzyme for 5 min
at 30C before the substrate was added The control reactions received solvent alone No effect of the solvents on epoxide or diol partitioning between the organic and aqueous phases was found
R E S U L T S
Cloning of BNSEH1
An epoxide hydrolase clone was isolated from a cDNA library constructed from B napus leaves treated for 5 h with MeJa The library average insert size was 1.3 kb and the isolated BNSEH1 clone contained 1273 base pairs The cDNA was sequenced in both directions and contained 230 base pairs of untranslated 3¢-sequence but was observed to lack the 5¢-end of the coding sequence A 5¢-RACE PCR on total RNA uncovered the initiator methionine codon and the few downstream residues missing, as well as 100 base pairs of the 5¢-upstream region including the transcription start site The products from the 5¢-RACE contained two ambiguities Five of the 18 clones sequenced differed from the cDNA by having a T instead of C at positions 145 and
249, respectively Neither of the two variants resulted in amino acid substitutions, however The predicted translated product of the cDNA contained 318 amino acids with the C-terminal tripeptide SKF, and lacked long hydrophobic regions (Fig 1)
Expression in yeast and purification of recombinant BNSEH1
The BNSEH1 was expressed intracellularly in yeast cells at high levels (£ 10% of the total protein and routinely
> 5 mgÆL)1) upon induction with methanol Several trans-formants were assayed in order to find highly expressed clones since multiple copies can be inserted in this system One highly expressed clone was found and used for all subsequent analysis The sEH activity in the cells increased rapidly after addition of methanol and cells were usually harvested after four days of culture After several passages
of the yeast cells through a French press the lysis efficiency reached 90–95% as determined by phase-contrast micro-scopy The crude extract was filtered, centrifuged and passed through a nickel resin with affinity for the histidine tag Fractions with more than 80% purity of BNSEH1 were used for the biochemical analysis
Trang 4Physico-chemical properties of recombinant BNSEH1
The mass of BNSEH1 was estimated as 37 kDa based on
SDS/PAGE with Coomassie staining (Fig 2A) The
theor-etical mass of BNSEH1 calculated from the predicted
amino acid sequence of the cDNA corresponds to
36 164 Da (37 096 Da with his-tag) assuming no
post-translational modifications The BNSEH1 over-expressed
enzyme cross-reacted with antibodies raised against AtsEH1
and the sizes of the sEHs were indistinguishable upon
Western blot analysis (Fig 2B) Twice the amount of
BNSEH1 protein relative to AtsEH1 was needed to reach
an equal signal intensity No reaction was observed when
samples were incubated with preimmune serum or when a
P pastorisextract without BNSEH1 was probed with the
specific antibodies
Gel filtration analysis of recombinant BNSEH1
The BNSEH1 was subjected to size exclusion
chromato-graphy to determine its oligomeric state BNSEH1 eluted as
a distinct single peak based on absorbance at 280 nm and
TSO activity (results not shown) The size of the native
BNSEH1 corresponded to 45 kDa
Biochemical characterization of recombinant BNSEH1 The recombinant enzyme had low activity towards CSO with values of 15 and 18 nmolÆmin)1Æmg protein)1for the two preparations tested TSO on the other hand served as a better substrate with at least twofold higher turnover Kinetic parameters were determined for the recombinant enzyme towards TSO at 30C Based on the Lineweaver-Burk rate equation, Vmax was determined to 0.46 and 0.48 lmolÆmin)1Æmg)1 in two experiments while the Km value was 11 lM in both cases (Fig 3) The kcat was calculated to 0.3 s)1 for TSO Substrate saturation was observed above 50 lMTSO The temperature optimum for the enzyme using TSO as substrate was found to be around
55C (Fig 4A) At this temperature a threefold higher activity was reached compared to standard conditions chosen at 30C The enzyme was almost completely inactivated at 64C and at higher temperatures The enthalpy of activation of the reaction was estimated from Arrhenius plots to 47 kJÆmol)1 The pH optimum for enzymatic TSO hydrolysis was found to be broad and around pH 6–7 (Fig 4B) At pH 4.5 almost 50% of maximum activity was obtained while activity dropped rapidly at higher alkaline pH The activity in potassium
Fig 1 Protein sequence alignment ( CLUSTALW 1.8) ofcloned plant epoxide hydrolases The terminal asterisks illustrate the suggested peroxisomal targeting signal The following sequences were retrieved for analysis from the GenBank database; B_napus (AJ459780), A_thaliana (D16628), G_max (CAA55294), S_tuberosum (U02497), E_lagascae 1 (AF482450), N_tabacum (AAB02006) The predicted catalytic residues are bolded and indicated
by * in the alignment Identical and similar residues are shown with black and grey backgrounds, respectively.
Trang 5phosphate buffer at pH 6–6.5 was similar to Tris/HCl
buffer at pH 7 It was noted also for the Arabidopsis
recombinant sEH that enzyme activity was higher in Tris
buffer compared to potassium phosphate at the same pH
[26] Apparent pKavalues for the active site were calculated
to 6.1 and 7.4, probably corresponding to the histidine and
tyrosine residues, respectively [6–8]
Effects of inhibitors on recombinant BNSEH1 enzyme activity
Several compounds were tested for potential inhibitory effects on the hydrolysis of TSO by the recombinant enzyme (Fig 5) N,N¢-dicyclohexylcarbodiimide was found to be a potent inhibitor and 10 lM of this compound abolished enzyme activity The corresponding N,N¢-dicyclohexylurea caused 30% inhibition at 10 lM and almost abolished activity at 1 mM The sulfhydryl reagent parahydroxy-mercuribenzoate and the histidine-reactive x-bromo-4-nitro-acetophenone gave intermediate inhibition (Fig 5A) The solvent tetrahydrofuran was found to cause a linear decrease in activity at increasing concentrations of the solvent (Fig 5B) Quercitin and chalcone oxide had weaker effects but caused 75% and 98% inhibition at 1 mM
concentration, respectively b-Naphtoflavone had no effect
up to 100 lMbut caused 50% inhibition at 1 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide had no effect on the enzyme activity up to 1 mM b-naphtoflavone, quercitin and N,N¢-dicyclohexylcarbodiimide did not dissolve completely
at 1 mM final concentration so the free concentration is actually lower Addition of ethanol alone had only a minor effect on the enzyme activity while addition of dimethyl-sulfoxide caused almost 20% reduction in activity and the solvent control was set to 100%
Fig 3 Kinetic analysis ofrecombinant BNSEH1 Enzyme activity was
determined using TSO at different concentrations from 0.98 to
187.5 l M in 0.1 M potassium phosphate, pH 7, using 0.1 lg of
BNSEH1 enzyme A Lineweaver-Burk plot used to calculate the
kin-etic parameters is shown based on data from one out of two
repre-sentative experiments.
Fig 4 Effects oftemperature and pH on recombinant BNSEH1 enzyme activity (A) Enzyme activity was measured using 100 l M TSO in 0.1 M
potassium phosphate buffer (pH 7) at different temperatures during
11 min using 0.1 lg of BNSEH1 enzyme Results from two experi-ments (j and d symbols, respectively) are shown (B) Enzyme activity was measured using 100 l M TSO in 0.1 M potassium phosphate buffers (pH 4.5–7) (d,s), 0.1 M Tris/HCl buffers (pH 7–9) (j,h) or 0.1 M glycine buffers (pH 9–11) (m,n) using 0.1 lg of BNSEH1 enzyme Results from two experiments are shown.
Fig 2 Electrophoretic analysis ofrecombinant BNSEH1 (A) SDS/
PAGE was performed using 12.5% acrylamide gels The samples
analysed were molecular mass standard (lane 1), Pichia extract (lane 2)
and recombinant BNSEH1 enzyme purified on nickel-agarose resin
(1.5 lg protein) with sizes as indicated in kDa to the left (B) Western
blot analysis using antibodies raised to recombinant sEH1 from
A thaliana (AtsEH1) The samples analysed were purified
recombin-ant AtsEH1 enzyme (lane 1), purified recombinrecombin-ant BNSEH1 enzyme
(lane 2) and Pichia extract (lane 3) The molecular mass standard with
sizes indicated in kDa is to the left.
Trang 6D I S C U S S I O N
The BNSEH1 was cloned and found to encode a protein of
318 amino acid residues The recombinant enzyme was
functional and readily detected by assay of TSO hydrolysis
in yeast extracts upon over-expression in P pastoris using a
methanol inducible promoter [27] The his-tagged BNSEH1
was obtained at > 80% purity after one-step purification
on Ni-agarose and the subunit mass could be determined to
37 kDa No misfolding seemed to occur since all protein
fractions recovered from the affinity chromatography
displayed activity and a symmetrical active peak was
obtained upon gel filtration analysis of the native enzyme
Of the plant sEH sequences available in the database,
BNSEH1 is most closely related to the Arabidopsis AtsEH1
with 85% identity in the predicted amino acid sequence
Identity to the other plant sEHs was in the range 50 to 60%
with a higher similarity to soybean (Glycine max) and
potato than to tobacco and E lagascae sEH (Fig 1) The
five catalytic residues of sEH in plants and mammals [9] are
conserved also in BNSEH1 suggesting that the properties
are similar to the potato and Arabidopsis enzymes [4]
Gel filtration analysis showed that the recombinant
BNSEH1 is a monomer The apparent native mass of
45 kDa is slightly higher than expected for a monomeric
BNSEH1 but can be due to an altered diffusion in the gel matrix because of the histidine tag Potato and Arabidopsis AtsEH1 were also shown to be monomers [4] while a soybean sEH was reported to be a dimer [12] An obvious difference between the soybean and the other plant sEH is
an N-terminal extension of 25 amino acids (Fig 1) The function of these residues is not clear but it is tempting to assign to it a role for dimerization The mammalian sEHs have a N-terminal extension of 250 amino acids, which the plant enzymes lack It has been proposed that this extension, containing a proline-rich sequence of 30 amino acids, is capable of dimerization transition [6] These prolines are not present in the soybean N-terminal (Fig 1) In mammals, a conserved signal sequence of three amino acids in the C-terminus (PTS1) is necessary and sufficient to direct proteins to peroxisomes [28] Further work is necessary to determine if the C-terminus, SKF, as found in BNSEH1 and AtsEH1, is a true peroxisomal targeting signal (PTS1)
in these plants
The BNSEH1 is active towards TSO but much less active towards the CSO isomer This substrate pair thus seems to give similar results on oilseed rape sEH as earlier shown also for the Arabidopsis and potato enzymes [4] The efficiency is lower for the oilseed rape enzyme compared to the Arabi-dopsisenzyme, AtsEH1, expressed in P pastoris [26] The Arabidopsisenzyme had a Vmaxof 2 lmolÆminÆmg)1and Km
around 5 lMfor TSO Also the CSO rate was higher for the Arabidopsisenzyme Unfortunately kinetic analyses of these enzymes towards CSO are hampered of the low solubility of the substrate at higher concentrations Morisseau et al [23] reported that carboxylate modifying agents such as N,N¢-dicyclohexylcarbodiimide and its hydrolysis product, N,N¢-dicyclohexylurea, showed strong inhibition of mam-malian sEH using 4-nitrophenyl-trans-2,3-epoxy-3-phenyl-propyl carbonate or 1,3-diphenyl-trans-propene oxide as substrates Also the B napus sEH, using TSO as substrate, was inhibited by these carboxylate modifying compounds (Fig 5), most probably through interference with the activating tyrosine residues [29] Chalcone oxides that originally were reported as potent inhibitors for mammalian sEH [30] does not seem to be very efficient on plant sEHs including AtsEH1 [4] and BNSEH1 Recent experiments with the soybean sEH, using the substrate 9,10-epoxystearic acid, suggested that also the enantioselectivity differs between plant and mammalian sEHs [31]
The BNSEH1 clone was isolated from a cDNA library prepared from MeJa-treated leaves sEH has been described
to be up-regulated at the transcript level by MeJa in potato [11] but in Vicia sativa seedlings sEH activity remained unaffected by MeJa treatment [32] No MeJa induction of sEH transcripts was observed in B napus seedlings kept in hydroponic cultures (S Bellevik, F Sitbon & J Meier) suggesting that BNSEH1 probably has a constitutive expression The only plant hormone besides MeJa reported
to induce sEH is auxin, which increased transcript levels of a sEH in Arabidopsis within 1 h of treatment [14] Analysis of such experiments must keep in mind that sEHs recently have been identified in multiple copies in several plants Preliminary Southern blot data suggest that at least four epoxide hydrolase genes are present in B napus (S Bellevik,
J Lin & J Meier) In Arabidopsis, the known and putative epoxide hydrolase genes inferred by data mining of the genome sequence [22] are intrinsically divergent
Fig 5 Effects ofinhibitors on recombinant BNSEH1 enzyme activity.
Enzyme activity was determined using 100 l M TSO in the presence of
various inhibitors in 0.1 M potassium phosphate, pH 7, using 0.1 lg
BNSEH1 enzyme The inhibitors tested were (A)
sodium-parahyd-roxymercuribenzoate (s), x-bromo-4-nitroacetophenone (m),
N,N¢-dicyclohexylcarbodiimide (j), and N,N¢-dicyclohexylurea (d)
(B) tetrahydrofuran The solvent control was set to 100% activity.
Trang 7(S Summerer & J Meier) We have over-expressed cDNAs
and measured activity on three of the Arabidopsis genes so
far, proving that several predicted isoforms are functional
(S Summerer & J Meier) The BNSEH1 sequence is highly
similar to AtsEH1 with an even distribution of mismatches
throughout the alignment (Fig 1) The homology is also
reflected in the cross-reaction of BNSEH1 with antibodies
specific to the AtsEH1 (Fig 2) Arabidopsis and oilseed rape
belong to the same family, Brassicaceae, and extensive
synteny and multiplicated genome segments seem to be
common [33] B napus is amphidiploid and the number of
sEH genes can be expected to be higher than for
Arabid-opsis When the B napus and Arabidopsis sEH gene families
are further characterized it will become possible to
deter-mine a more specific gene relationship between these species
Oilseed rape is the third largest oil crop in the world and
there are potential industrial applications for transgenic
plants modified for enzymes such as epoxide hydrolases
For example, overproducing oxylipins in the vegetative
tissues resulting in a better endogenous biotic stress
protection would be useful for the agriculture and
environ-ment in terms of reduced pesticide spraying and increased
crop yields A modified lipid composition in the seeds of
B napus is also interesting as a renewable source for
technical oils [19] Knowledge of the function and regulation
of epoxide hydrolase isoforms in plants open up possibilities
for future engineering to redirect fatty acid metabolism into
the desired products
A C K N O W L E D G E M E N T S
This work was supported by grants from the Foundation for Strategic
Research We are grateful to Mikael Widersten, Uppsala University,
for discussions on enzyme kinetics.
R E F E R E N C E S
1 Oesch, F (1973) Mammalian epoxide hydrases: inducible enzymes
catalysing the inactivation of carcinogenic and cytotoxic
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