Molecular Cloning and Characterization of 12-Oxophytodienoate Reductase, an Enzyme of the Octadecanoid Signaling Pathway fromArabidopsis thaliana

Using partial amino acid sequence information for 12-oxophytodienoate-10,11-reductase obtained from Corydalis sempervirens we have cloned the homologous enzyme fromArabidopsis thaliana. The open reading frame of the cDNA encodes a polypeptide of 372 amino acids (M r = 41,165) with significant similarity to the sequence of Old Yellow Enzyme from Saccharomyces carlsbergensis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem.266, 20720–20724), a flavin (FMN)-protein catalyzing the NADPH-dependent reduction of the olefinic bond of α,β-unsaturated carbonyls. Specifically, all residues required for binding of FMN in Old Yellow Enzyme are conserved in the A. thaliana sequence, as are all residues associated with catalytic activity. The enzyme was functionally expressed from its cDNA inEscherichia coli and thus proven to encode OPDA reductase. Further similarities of OPDA reductase and yeast Old Yellow Enzyme include their binding to and elution by reductant fromN-(4-hydroxybenzoyl)aminohexyl-Sepharose, the immunoreactivity of yeast Old Yellow Enzyme with an antiserum raised against plant OPDA reductase and the demonstration that Old Yellow Enzyme is an active OPDA reductase. It is thus conceivable that the physiological role of Old Yellow Enzymes now known from bacteria, yeasts, and higher plants, is in oxylipin metabolism.

Octadecanoids have multiple physiological functions in plants (2). They are signal transducers in herbivore defense (3,4), pathogen defense (5), mechanotransduction (6,7), they promote senescence (8) and induce leaf defense volatile emission (9,10). Only recently, evidence has emerged that different octadecanoids may have distinct and different profiles of biological activities. For example, OPDA is the strongest inducer of tendril coiling in Bryonia dioica, a process of mechanotransduction (11), while mainly jasmonic acid is held responsible for inducing herbivore and pathogen defenses (3,5), whereas conjugates of jasmonic acid with amino acids seem to be inducers of plant volatile emission (12). The molecular analysis of these processes has only begun.
Little is known about the mechanisms regulating octadecanoid production and translocation within a tissue. The biosynthetic pathway from ␣-linolenic and to jasmonic acid (1) in all probability involves three compartments. OPDA is synthesized in plastids (13)(14)(15). In support of this, suppression of expression of the only plastidic lipoxygenase isoform occurring in Arabidopsis thaliana by antisense technology suppressed jasmonate accumulation (15), and allene oxide synthase genes cloned from flax (16) and A. thaliana (14) encode precursor polypeptides with transit peptides characteristic for plastid targeting sequences. On the other hand, ␤-oxidation in plants occurs exclusively in microbodies (peroxisomes and glyoxisomes). Thus, this should be the compartment where OPC-8:0 is being converted to jasmonic acid.
OPDA reductase plays a central role in octadecanoid biosynthesis in that the enzyme controls metabolite flow from the C18 group to the C12 group of cyclic ␣-linolenic acid derivatives. The enzyme is soluble and not associated with either plastids or microbodies and is thus probably cytosolic (17). This location reinforces the notion that OPDA reductase is a critical checkpoint in octadecanoid biosynthesis. To understand the molecular aspects of cellular control over OPDA reductase and of pathway control by OPDA reductase, we have recently isolated the enzyme in pure form (17) (EC 1.3.1.42) to make it accessible to reverse genetics. Here, we report the molecular cloning of OPDA reductase from A. thaliana and its functional expression in a bacterial host. Sequence analysis has revealed a surprising similarity of OPDA reductase to Warburg's Old Yellow Enzyme (OYE) (18,19) (EC 1.6.99.1). This prompted a more detailed comparison of the two enzymes. We report that yeast OYE also has OPDA reductase activity. It is thus probable that the, as yet unknown, biological function of OYE may involve reduction of ␣,␤-double bonds in cyclic oxo-fatty acids.

EXPERIMENTAL PROCEDURES
Purification and Protein Sequencing of OPDA Reductase-OPDA reductase was purified in enzymatically active form from cell suspension cultures of Corydalis sempervirens as described previously (17). The protein (25 mg) was finally precipitated with methanol at Ϫ20°C and subjected to SDS-PAGE (20) on a preparative 12% gel from which the 41-kDa band was electroeluted using a Biotrap BT1000 (Schleicher & Schuell), precipitated with methanol, and redissolved in 1 ml of double distilled water. Peptides were generated by cyanogen bromide cleavage at Met-X bonds according to Smith (21) and separated by SDS-PAGE according to Schä gger and von Jagow (22). They were then electrotransferred to polyvinylidene difluoride membranes (Immobilon TM , Millipore) at 50 V (20°C, 30 min) in 10 mM CAPS buffer, pH 11.0, containing 10% (by volume) methanol, and localized with Coomassie Brilliant Blue. Exised bands were subjected to gas phase Edman sequencing (performed by Dr. J. Block, Max-Planck-Institut fü r Molekulare Physiologie, Dortmund).
Molecular Identification and Nucleotide Sequencing of OPDA Reductase-The amino acid sequence information obtained for the C. sempervirens OPDA reductase was used to screen data bases of expressed sequence tags of A. thaliana. A 33-amino acid sequence stretch aligned with high similarity (Ͼ78% identical amino acids) to EST clone 179A13T7 of the NCBF EST-clone collection. This clone contained a 1.3-kilobase pair insert which was sequenced on both strands employing the dideoxy chain termination method (23) and series of subclones exonuclease III-digested from either the 5Ј-or the 3Ј-end of the cDNA (24). The region between nucleotides 800 and 950 was additionally sequenced using specific primers corresponding to nucleotides 990 -970 and 725-745.
Two primers were designed from the 3Ј-and 5Ј-ends of EST clone 179A13T7 to allow amplification of the entire coding sequence of the putative OPDA reductase from a cDNA library of A. thaliana, race Columbia, shoots maintained in ZAP (provided by Dr. R. Hell, Bochum). Primer A (5Ј-GCT CAA GCT TGG ATC CAT GGA AAA CGG AGA AGC-3Ј) encompassed the translational start ATG and nucleotides 75-88 of the sequence of EST clone 179A13T7 flanked by a BamHI restriction site (underlined), primer B (5Ј-GAG GGC TGC ACC CGG GGG AAA CAC-3Ј) encompassed nucleotides 1217-1225 of the EST sequence flanked by a SmaI site (underlined). Using these primers, a cDNA fragment of the predicted size (1.18 kilobase pairs) was obtained. The amplified fragment was cloned into the vector pBS SK(ϩ) (Stratagene, La Jolla, CA) and sequenced on both strands to provide inde-pendent verification of the nucleotide sequence obtained for the EST clone 179A13T7. Furthermore, the polymerase chain reaction-generated full-length A. thaliana cDNA fragment was cloned into the protein expression vector pQE-30 (Qiagen) using the BamHI/SmaI restriction sites and transfected into Escherichia coli strain XL1-blue (25) by electroporation (26) for protein expression studies. Nucleotide sequences were aligned using AssemlyLIGN TM (Eastman Kodak Co.), amino acid sequences were aligned using DNAsis (Hitachi) and MacVector (Kodak) software.
Protein Expression in E. coli-Expression of protein was induced by adding isopropyl-1-thio-␤-D-galactopyranoside (IPTG) to log-phase bacteria up to 2 mM concentration for 5 h. In parallel, control bacteria transfected with pQE-30 containing no insert, were processed. Native, hexahistidine-tagged protein was purified from bacterial lysates on Ni-nitrilotriacetate (Ni-NTA) resin according to the protocol (no. 5) provided by the supplier (Qiagen) and was obtained as a 1 mg/ml solution in 50 mM Na-phosphate, 300 mM NaCl, 10% (v/v) glycerol, 500 mM imidazole, pH 6.0. The eluate was further concentrated using Amicon Centriprep-10 ultrafiltration units and taken up in 0.1 M Tris-HCl, 0.1 M (NH 4 ) 2 SO 4 , pH 8.0 (1.2 mg/ml protein). This fraction was used for affinity chromatography.
Affinity Chromatography on N-(4-Hydroxybenzoyl)aminohexyl-Sepharose-OYE was purified from bakers' yeast (Saccharomyces cerevisiae, 500 g of packed cells) as described elsewhere (27). The synthesis of N-(4-hydroxybenzoyl)aminohexyl (HBA)-Sepharose followed the protocol of Abramovitz and Massey (27) and Stott et al. (28). The affinity matrix (10 ml) was equilibrated with 0.1 M Tris-HCl, 0.1 M (NH 4 ) 2 SO 4 , 10 M phenylmethylsulfonyl fluoride, pH 8.0, and used at 4°C. Total soluble yeast protein representing 500 g of cells was applied at a flow rate of 1.5 ml/min. OYE was eluted with the equilibration buffer containing 3 mM sodium dithionite (flow rate, 1.5 ml/min) and concentrated, using an Amicon Centriprep-10 ultrafiltration unit, to a protein concentration of 1.5 mg/ml. Prepurified protein fractions from C. sempervirens cell cultures (17) (2 mg) or Ni-NTA affinity purified recombinant protein from cell lysates of E. coli (1-2 mg) (see above) was subjected to affinity chromatography on HBA-Sepharose under the same conditions.
Assays for OPDA Reductase Activity-The substrate, cis-(Ϯ)-OPDA was synthesized enzymatically from 13-hydroperoxylinolenic acid using recombinant A. thaliana allene oxide synthase (29). The trans-isomer was produced from the cis-isomer by base-catalyzed enolization (30). Assays contained, in a total volume of 0.5 ml or 1.0 ml of buffer (50 mM potassium phosphate, pH 7.5) 0.1 mM substrate, 1.0 mM NADPH, and protein (10 g of partially purified, recombinant OPDA reductase or control protein (data in Fig. 5C); 1 g of affinity-purified OPDA reductase (data in Fig. 5F); 2 g of partially purified OYE (data in Fig. 7)) and were incubated for 30 -60 min at 25°C (reaction linear under these conditions for 60 min). Because of potential NADPH-oxidase side reactions, all enzyme activities were based on quantitations of product formed versus substrate consumed made by gas chromatography-mass spectrometry as detailed in Schaller and Weiler (17). Product verification was further done by using 17[ 2 H 2 ],18[ 2 H 3 ]OPDA as substrate and gas chromatography-mass spectrometry analysis of the reaction products (17).
Immunological Methods-Proteins were separated by SDS-PAGE (20) and electroblotted (31) to nitrocellulose membranes. Membranes were then incubated at room temperature for 1 h in TBS-T buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1.0 mM MgCl 2 , 0.05% (v/v) Tween 20) containing 2% skim milk powder. The primary antibody against OPDA reductase from C. sempervirens (17) was diluted 1:10,000 in TBS-T buffer. Membranes were incubated in this solution for 1 h at room temperature and were then washed three times, 10 min each, in TBS-T buffer, followed by incubation with goat anti-rabbit IgG coupled to alkaline phosphatase (Promega) and enzymatic analysis as described previously (32).

RESULTS AND DISCUSSION
Partial Amino Acid Sequence of OPDA Reductase-To obtain partial amino acid sequence information, OPDA reductase (25 mg) was purified from cell suspension cultures of C. sempervirens and obtained as a soluble, yellow preparation containing predominantly a single, 41-kDa, polypeptide (17). Treatment with CNBr yielded, on SDS-PAGE using 16.5% separating gels, seven fragments ranging from 4 to 12 kDa in apparent molecular masses. N-terminal Edman gas-phase sequencing of the two predominant peptides yielded single sequences in each

12-Oxophytodienoate Reductase Related to Old Yellow Enzyme
case. These were: 9-kDa peptide, RKAFNGTFIAAGGYKKD-DGGKAAIENHHD, and 12-kDa peptide, GKFNLSHRVV-LAPLTFDRSYDNLPQQHAAAYYY. The latter sequence of 33 amino acids was used to screen protein data bases and A. thaliana EST data bases. The peptide aligned with significant similarity (73% identical plus similar amino acids) to an Nterminal stretch of the known sequence of S. cerevisiae OYE. One A. thaliana EST clone (179A13T7), partially sequenced from its 5Ј-end, was found to encode a protein clearly homologous (Ͼ78% identical residues in the 33-amino acid sequence stretch) to C. sempervirens OPDA reductase.
Sequencing and Characterization of the A. thaliana OPDA Reductase Homolog-The complete nucleotide sequence of EST clone 179A13T7 is shown in Fig. 2. The sequence encompasses an open reading frame starting at position 72 and ending at position 1187 that would encode a polypeptide of 41165 Da with a calculated pI of 6.35. Since the apparent molecular mass of purified OPDA reductase was found to be 41 kDa, the sequence is considered complete. This is further suggested by the 3Ј-noncoding region ending with poly(A) and by the fact that the first ATG and the sequence elements surrounding it are in agreement with the consensus motif for plant translational start sites (33). Also aligned in Fig. 2 are the two partial sequences obtained from amino acid sequencing of the purified OPDA reductase of C. sempervirens, both of which are clearly very similar and preceded by a methionine in the A. thaliana-deduced sequence, in agreement with the CNBr cleavage sites.
A comparison of the complete amino acid sequence deduced from the cDNA with sequence information already available in data bases (Fig. 3) revealed significant similarities with all yeast OYEs, namely OYE1 from S. carlsbergensis (19), OYE2 and OYE3 from S. cerevisiae (28,34) (OYE1 to OYE3 in Fig. 3), Schizosaccharomyces pombe (OYE A and B) and Kluyveromyces lactis (35) (KY E1 ) as well as with a recently cloned, bacterial morphinone reductase (36), which is more similar to the putative OPDA reductase than to OYEs and with the Candida albicans estrogen-binding protein (EBP1 in Fig. 3), which is an oxidoreductase (37).
More detailed analysis of the sequences strengthens the evidence that the protein encoded by EST clone 179A13T7 belongs to the OYE family of FMN-containing reductases (Fig. 4). From the crystal structure of OYE1 from S. cerevisiae, it has been deduced that the following amino acids (numbering as in Fox and Karplus (38)  is also the preferred electron donor of OPDA reductase (17). Furthermore, the turn between the ␤4-␣4 domains of OYE contains a characteristic structural signature of unknown function conserved among OYE subfamily members C-terminally adjacent the active site (amino acids 207-222 of OYE1 (39)). This stretch is also conserved between OYE and the A. thaliana protein (11 amino acids out of 16 are identical). OYE1 is a member of the ␣,␤-barrel class of proteins (38). Using the software Swiss-Model/RasMol v2.5 (40,41), the predictions of the most likely three dimensional structure of the A. thaliana protein yields an almost perfect fit to the three-dimensional structure of yeast OYE1 and clearly that of an ␣,␤-barrel protein. Thus, information from the cDNA sequence and from structure predictions (not shown) leaves little doubt that the protein encoded by EST clone 179A13T7 represents a plant homolog of OYE. No indication of putative targeting sequences or transit-peptide sequences can be derived from the deduced amino acid sequence, in agreement with biochemical data showing that OPDA reductase is, in all probability, a cytosolic enzyme (17).
Functional Expression of OPDA Reductase from E. coli-The polymerase chain reaction-generated cDNA encompassing the complete coding sequence of the putative OPDA reductase from A. thaliana, generated as detailed under "Experimental Procedures," was inserted, as a BamHI/SmaI restriction fragment, into the multiple cloning site of the protein expression vector  Fig. 3. CR-OYE, deduced amino acid sequence of a short partial cDNA of unknown function isolated from Chenopodium rubrum (46). Aligned using MacDNAsis. Consensus sequence: boldface capitals, residues conserved in all sequences; capital letters, conserved in all but one sequences; small letters, conserved in all but two sequences; *, amino acid difference specific to OPDA reductase; conserved substitutions: n, nonpolar; p, polar; b, basic; a, acidic. pQE-30. The construct (pQE-30-AT1) was electroporated into the E. coli host XL1-blue. IPTG-induced expression from this vector results in the formation of an N-terminally hexa-histidine (His 6 )-tagged protein. Although different concentrations of IPTG (0.1-2 mM), temperatures ranging from 28 to 37°C, and induction periods between 1 and 16 h were tested, under all conditions, most of the expressed protein was associated with inclusion bodies, and only a minor portion remained soluble (not shown). Thus, total soluble protein from lysates of cells transformed with pQE-30-AT1 was affinity-purified on Ni-NTA-agarose. For controls, protein from cells transfected with the empty vector pQE-30 was always processed in parallel. As can be seen from Fig. 5A, there was little difference in the polypeptide patterns from cells transfected with either pQE-30-AT1 or pQE-30 alone, even after Ni-NTA-agarose af-finity chromatography (Fig. 5A, lanes e versus E), since the expression level was too low to allow for effective competition of the tagged fusion protein with endogenous bacterial proteins for the metal binding sites on the column. The relevant area in the electropherograms was masked by a prominent ϳ43-kDa polypeptide that co-eluted on Ni-NTA-agarose. The successful expression of the desired 41-kDa protein, and its binding to the affinity matrix, was, however, unequivocally demonstrated by immunoblotting using an antiserum against the C. sempervirens enzyme (17) (Fig. 5B). This protein was absent in control bacteria. Although some reduction of the substrate, OPDA, was observed in crude soluble protein fractions from control (pQE-30 transfected) bacteria, the determination of OPDA reductase activity in the fraction eluting from Ni-NTA-agarose revealed a clear increase in OPDA reductase activity in the immunoreactive protein fraction obtained from pQE-30-AT1transfected cells (Fig. 5C; protein as in Fig. 5B, lane E versus lane e). For structural identification of the reaction product, see below (Fig. 7).
A characteristic property of yeast OYE is its specific retention on HBA-Sepharose and elution with the reductant, sodium dithionite (27). It was found that the recombinant OPDA reductase (affinity-prepurified on NTA-agarose) was also retained by this column and eluted by sodium dithionite (Fig. 5,  D and E). The experiment revealed some heterogeneity of the recombinant enzyme, a fast eluting fraction, which was an active OPDA reductase, a slower eluting, likewise enzymatically active fraction, and a strongly retained, immunoreactive polypeptide migrating slightly slower (43 kDa) in SDS-PAGE and being devoid of enzymatic activity. Clearly, OPDA reductase activity was associated with the recombinant 41-kDa polypeptide. The 43-kDa band probably represents reductase inactivated by some post-translational modification by the bacterial host cell, while the fast eluting, active reductase (E-1 fraction, Fig. 5, D-F) is front-end eluted upon contact with reductant under our fast flow conditions.
Next, it was determined whether the enzyme purified from C. sempervirens cell suspension cultures would also be retained on HBA-Sepharose. This was the case (Fig. 6, lanes 3-7); electrophoretically pure polypeptide was obtained by sodium dithionite elution (Fig. 6, lane 5). As a control, reference (27) yeast OYE was also purified (Fig. 6, lane 1) for further characterization. The yeast fraction was checked for its immunoreactivity with the OPDA reductase antiserum from the higher plant (Fig. 6, lane 2); there was a clear and specific decoration FIG. 5. A, Coomassie-stained polypeptides separated by SDS-PAGE (12% gel) and representing l and L, whole cell lysates; w and W, column effluents; and e and E, eluates from Ni-NTA affinity columns of bacterial protein obtained from E. coli XL1 blue cells transfected with pQE-30-AT1 (harboring the OPDA reductase cDNA; L, W, E) or transfected with pQE-30 (control plasmid without insert; l, w, e). B, immunoblot of the same fractions developed with antiserum raised against OPDA reductase from C. sempervirens (17). C, OPDA reductase activity in affinity (Ni-NTA)-purified fractions containing recombinant OPDA reductase (E, corresponding to protein shown in lanes E in part A and B of this figure) or control protein (e, corresponding to protein shown in lanes e in part A and B of this figure). In both cases, 10 g of protein were used under otherwise standard conditions. D, elution from HBA-Sepharose of protein from the Ni-NTA-prepurified fraction containing recombinant OPDA reductase (designated E in all other parts of this figure). The arrow denotes the start of the elution process. Fractions collected separately are numbered E-1 to E-6. E, immunoblot analysis and F, relative OPDA reductase activity in fractions E-1 to E-6 collected from the HBA-Sepharose affinity column. For enzyme assays, 1 g of protein from each fraction was used.  2, 6, and 7), an antiserum raised against purified OPDA reductase from C. sempervirens (17) was used.

12-Oxophytodienoate Reductase Related to Old Yellow Enzyme
of the 43-kDa polypeptide representing yeast OYE. Altogether, these results strengthen the conclusions drawn from the sequence information and prove that yeast OYE and higher plant OPDA reductase are similar in their size, overall biochemical properties, structure, and immunoreactivity and thus must be quite closely related. Similarities in enzymatic reactivity of OPDA reductase and OYE were suggested earlier by the observation that OPDA reductase would convert 2-cyclohexenone, a standard OYE substrate (17).
Yeast Old Yellow Enzyme Has OPDA Reductase Activity-Although being the first discovered flavoprotein (18), the biological function of Warburg's Old Yellow Enzyme is still uncertain (42). The enzyme catalyzes the oxidation of a range of cyclic ␣,␤-unsaturated carbonyl compounds to phenols with concurrent reduction of the olefinic bond of a second substrate molecule, while also catalyzing the NADPH-dependent reduction of the olefinic bond in other cyclic or acyclic ␣,␤-unsaturated carbonyl compounds, e.g. (42). A natural substrate, however, is not known. We tested, if OPDA was a substrate for OYE. To avoid any potential contamination by endogenous materials, racemic pentadeuterated cis-OPDA (18[ 2 H 3 ],17[ 2 H 2 ]-cis-(Ϯ)-12-oxophytodienoic acid) was used as a substrate for yeast OYE. As a control, recombinant A. thaliana OPDA reductase was analyzed in parallel. Both enzymes converted, in an NADPH-dependent reaction, the pentadeuterated substrate to a product that had identical retention times on high performance liquid chromatography and capillary gas chromoatography as well as identical mass spectra which were in complete agreement with those of authentic OPC-8:0 (not shown). Thermal isomerization converted both reaction products into their trans-isomers which, again, exhibited identical chromatographic and mass spectroscopic behavior (Fig. 7) in agreement with the literature (17). This established the structure of the reaction product unequivocally as [ 2 H 5 ]-OPC-8:0. Thus, yeast OYE can function as an OPDA reductase. Prostaglandin-like metabolites have been found in some yeasts (43,44) and were assigned a biological role in ascospore development (43,45). It is thus conceiveable that yeast OYE may play a role in the metabolism of yeast oxylipins.
Conclusions-OPDA reductase catalyzes a decisive step in biosynthesis of plant octadecanoids, signal transducers in mechanotransduction, pathogen, and herbivore defense. Molecular cloning has revealed that the enzyme is the first plant member of the OYE family of reductases that now comprises members in bacteria, fungi, and higher plants. While all OYEs catalyze the NADPH-dependent reduction of olefinic bonds of ␣,␤-unsaturated carbonyls, such as in morphinone (36), 4,4dimethyl-2-cyclohexenone (42), or 2-cyclohexenone (34,42), OPDA reductase is the only member within this family whose biological function is currently known. The proof that yeast OYE also has OPDA reductase activity suggests a physiological function of OYEs in oxylipin metabolism.

FIG. 7. Analysis of reaction products obtained from incubations of purified yeast OYE and of recombinant A. thaliana
OPDA reductase with pentadeuterated cis-(؎)-OPDA. Reaction products were extracted from the enzyme assay mixtures after 60 min of incubation at 30°C, converted into the trans-isomers and methyl esters and then analyzed by capillary GC-MS (17). Shown are A, total ion current (TIC) trace of the reaction products obtained with purified OYE (2 g of protein, otherwise standard conditions) and B, TIC trace of the reaction products obtained with purified recombinant A. thaliana OPDA reductase expressed in E. coli (10 g of protein, otherwise identical conditions). The trans-OPDA methyl ester eluted at t ϭ 831 s, trans-OPC-8:0 methyl ester eluted at t ϭ 800 s. C, electron impact (70 eV) mass spectrum of the reaction product (t ϭ 800 s) obtained with OYE; D, electron impact (70 eV) mass spectrum of the reaction product (t ϭ 800 s) obtained with OPDA reductase and fragmentation pattern of pentadeuterated OPC-8:0 methyl ester.