Báo cáo Y học: Brassica napus soluble epoxide hydrolase (BNSEH1) Cloning and characterization of the recombinant enzyme expressed in Pichia pastoris docx

Brassica napus soluble epoxide hydrolase (BNSEH1) Cloning and characterization of the recombinant enzyme expressed in Pichia pastoris Stefan Bellevik 1 , Jiaming Zhang 2 and Johan Meijer 1 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-stilbene oxide as substrate with V max of 0.47 lmolÆminÆmg )1 , K m of 11 l M and k cat of 0.3 s )1 . The optimum temperature of the recombinant enzyme was 55 °C 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) Eur. J. Biochem. 269, 5295–5302 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03247.x epoxide 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. EXPERIMENTAL PROCEDURES 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 37 °C for 16 h to form the corresponding urea (N,N¢-dicyclohexylurea) derivative [23]. Cloning of B. 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 pBluescript. The probe was random- primed using [ 32 P]dCTP (Amersham International, Bucks, England), purified by gel filtration and used at  10 6 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) ASMART TM RACE 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 using one nested primer pair after the first cDNA synthesis. In the first strand synthesis Superscript II TM Rnase 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, 65 °C 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 Prism TM 377XL DNA sequencer basedonaDYEnamic TM ET 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 in P. 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/OD cell suspension) using the [ 3 H]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 5296 S. Bellevik et al. (Eur. J. Biochem. 269) Ó FEBS 2002 3000 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 m M Tris/HCl, 40 m M imidazole, 500 m M NaCl, 1m M benzamidine, 1 m M phenylmethanesulfonyl fluoride, 20 m M 2-mercaptoethanol, pH 7) and cells ruptured by at least three passages through a 40K FrenchÒ pressure cell press (SLM Aminco, Urbana, IL) prior to centrifugation at 12 000 g for 10 min at 4 °C. The supernatant was sterile filtrated using 1.2 lm and 0.45 lm filters. HiTrap TM Chelating columns (Amersham Biosciences) were loaded with 0.1 M NiCl prior to addition of sample. The column was washed with start buffer and the enzyme eluted with 150 m M 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 0.1 M potassium phos- phate pH 7.2, 1 m M 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 [ 3 H]TSO or [ 3 H]CSO synthesized as described [3]. The routine assays contained 100 l M final substrate concentra- tion in 0.1 M potassium phosphate, pH 7.0 and were run at 30 °C 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). pK a values were obtained from intersections of asymptotes to the curve plot of log(k cat ) vs. pH. Activation enthalpy was calculated from Arrhenius plots of ln(k cat )vs.1/T based on values up to the denaturation point (55 °C). For the kinetic param- eters a range of approximately 10-fold higher and lower substrate concentrations of the predicted K m was 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 K m and V max values. When inhibitors were tested the inhibitor was preincubated with enzyme for 5 min at 30 °C 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. RESULTS 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. Ó FEBS 2002 Recombinant Brassica napus epoxide hydrolase (Eur. J. Biochem. 269) 5297 Physico-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. pastoris extract 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 )1 Æmg protein )1 for 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 30 °C. Based on the Lineweaver- Burk rate equation, V max was determined to 0.46 and 0.48 lmolÆmin )1 Æmg )1 in two experiments while the K m value was 11 l M in both cases (Fig. 3). The k cat was calculated to 0.3 s )1 for TSO. Substrate saturation was observed above 50 l M TSO. The temperature optimum for the enzyme using TSO as substrate was found to be around 55 °C (Fig. 4A). At this temperature a threefold higher activity was reached compared to standard conditions chosen at 30 °C. The enzyme was almost completely inactivated at 64 °C 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) of cloned 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. 5298 S. Bellevik et al. (Eur. J. Biochem. 269) Ó FEBS 2002 phosphate 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 pK a values 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 l M of this compound abolished enzyme activity. The corresponding N,N¢-dicyclohexylurea caused 30% inhibition at 10 l M and almost abolished activity at 1 m M . 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 m M concentration, respectively. b-Naphtoflavone had no effect up to 100 l M but caused 50% inhibition at 1 m M . 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide had no effect on the enzyme activity up to 1 m M . b-naphtoflavone, quercitin and N,N¢-dicyclohexylcarbodiimide did not dissolve completely at 1 m M 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 of recombinant 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 lgof 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 of temperature 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 lgofBNSEH1enzyme.Resultsfromtwoexperi- 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)using0.1lgofBNSEH1 enzyme. Results from two experiments are shown. Fig. 2. Electrophoretic analysis of recombinant 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 recombinant BNSEH1 enzyme (lane 2) and Pichia extract (lane 3). The molecular mass standard with sizes indicated in kDa is to the left. Ó FEBS 2002 Recombinant Brassica napus epoxide hydrolase (Eur. J. Biochem. 269) 5299 DISCUSSION 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- dopsis enzyme, AtsEH1, expressed in P. pastoris [26]. The Arabidopsis enzyme had a V max of 2 lmolÆminÆmg )1 and K m around 5 l M for TSO. Also the CSO rate was higher for the Arabidopsis enzyme. 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 of inhibitors 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. 5300 S. Bellevik et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (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 sEHgenescanbeexpectedtobehigherthanforArabid- 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. ACKNOWLEDGEMENTS 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. REFERENCES 1. Oesch, F. 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Brassica napus soluble epoxide hydrolase (BNSEH1) Cloning and characterization of the recombinant enzyme expressed in Pichia pastoris Stefan Bellevik 1 , Jiaming Zhang 2 and Johan Meijer 1 1 Department. putative epoxide hydrolase genes inferred by data mining of the genome sequence [22] are intrinsically divergent Fig. 5. Effects of inhibitors on recombinant BNSEH1 enzyme activity. Enzyme activity was. 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