Molecular Cloning of a Divinyl Ether Synthase IDENTIFICATION AS A CYP74 CYTOCHROME P-450*

Lipoxygenase-derived fatty acid hydroperoxides are metabolized by CYP74 cytochrome P-450s to various oxylipins that play important roles in plant growth and development. Here, we report the characterization of a Lycopersicon esculentum (tomato) cDNA whose predicted amino acid sequence defines a previously unidentified P-450 subfamily (CYP74D). The recombinant protein, expressed in Escherichia coli , displayed spectral properties of a P-450. The enzyme efficiently metabolized 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid but was poorly active against the corresponding 13-hydroperoxides. Incubation of recombinant CYP74D with 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid yielded divinyl ether fatty acids (colneleic acid and colnelenic acid, respectively), which have been implicated as plant anti-fungal toxins. This represents the first identification of a cDNA encoding a divinyl ether synthase and establishment of the enzyme as a CYP74 P-450. Genomic DNA blot analysis revealed the existence of a single divinyl ether synthase gene located on chromosome one of tomato. In tomato seedlings, root tissue was the major site of both divinyl ether synthase mRNA accumulation and enzyme activity. These results indicate that developmental expression of the divinyl ether synthase gene is an important determinant of the tissue specific synthesis of divinyl ether oxylipins.

Lipoxygenase-derived fatty acid hydroperoxides are metabolized by CYP74 cytochrome P-450s to various oxylipins that play important roles in plant growth and development. Here, we report the characterization of a Lycopersicon esculentum (tomato) cDNA whose predicted amino acid sequence defines a previously unidentified P-450 subfamily (CYP74D). The recombinant protein, expressed in Escherichia coli, displayed spectral properties of a P-450. The enzyme efficiently metabolized 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid but was poorly active against the corresponding 13-hydroperoxides. Incubation of recombinant CYP74D with 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid yielded divinyl ether fatty acids (colneleic acid and colnelenic acid, respectively), which have been implicated as plant anti-fungal toxins. This represents the first identification of a cDNA encoding a divinyl ether synthase and establishment of the enzyme as a CYP74 P-450. Genomic DNA blot analysis revealed the existence of a single divinyl ether synthase gene located on chromosome one of tomato. In tomato seedlings, root tissue was the major site of both divinyl ether synthase mRNA accumulation and enzyme activity. These results indicate that developmental expression of the divinyl ether synthase gene is an important determinant of the tissue specific synthesis of divinyl ether oxylipins.
Oxylipins comprise a group of bioactive compounds that are produced in plants from oxidative metabolism of polyunsaturated fatty acids. Several aspects of the function and biosynthesis of these compounds are analogous to the eicosanoid family of lipid regulators in vertebrates (1,2). Interest in plant oxylipins has focussed largely on jasmonic acid, a potent regulator of defense-related and developmental processes. The physiological role of jasmonic acid is well understood, and genes encoding all of the biosynthetic enzymes have been cloned (3)(4)(5). Much less is known about the biological function of other members of the plant family of oxylipins, which includes hydroxy, epoxyalcohol, keto, and divinyl ether derivatives (6 -8), and few of the genes encoding the key biosynthetic enzymes have been identified.
The biosynthesis of most plant oxylipins is initiated by lipoxygenases (LOXs) 1 that add molecular oxygen to either the 9 or 13 position of linolenic or linoleic acid (see Fig. 1). Hydroperoxide products of LOX are then committed to various branches of oxylipin metabolism. For example, allene oxide synthase (AOS) transforms 13-hydroperoxy linolenic acid  to an unstable allene oxide that either converts spontaneously to ␣and ␥-ketols or serves as the precursor for enzymatic synthesis of jasmonic acid and related cyclopentenones (9,10). A second well studied enzyme, hydroperoxide lyase (HPL), cleaves 13-HPOT into short chain aldehydes that contribute to the characteristic odor of fruits, vegetables, and green leaves (11). HPL-derived oxylipins are also implicated as anti-microbial toxins (12) and as signals for the regulation of growth properties and gene expression (13,14).
AOS and HPL are members of a family (CYP74) of cytochrome P-450s that are specialized for the metabolism of fatty acid hydroperoxides (15,16). In contrast to P-450 monooxygenases that require molecular oxygen and NADPH-dependent cytochrome P-450 reductase for activity, AOS (CYP74A) and HPL (CYP74B and C) utilize an acyl hydroperoxide both as the oxygen donor and as the substrate in which the new carbonoxygen bonds are formed. Consistent with this, AOS and HPL do not require O 2 or NADPH for activity and have reduced affinity for CO (17)(18)(19)(20). These catalytic features are shared by prostacyclin synthase and thromboxane synthase, two other P-450s that catalyze the rearrangement of C20 acyl peroxides in the arachidonic acid cascade (21). A better understanding of the CYP74 family of enzymes may thus provide general insight into the synthesis and function of fatty acid-derived signals in both plants and animals.
in green leaves of meadow buttercup (24,25). The structure of divinyl ether fatty acids in the marine alga Laminaria sinclairii suggests the involvement of a DES that metabolizes 13-and 15-hydroperoxides of C18 and C20 polyunsaturated fatty acids (26). Polyneura latissima, a red alga, accumulates an eicosanoid divinyl ether whose structure and coexistence with 9-hydroxy-eicosatetraenoic acid suggest a biosynthetic route from 9-LOX (27). The function of divinyl ether oxylipins in biological systems remains largely unknown. However, a recent report provided evidence that CA and CnA play a role in plant defense against the pathogenic fungus Phytophthora infestans (28).
Although much is known about the biosynthesis of divinyl ether fatty acids in various plant tissues, purification of DES or cloning of a DES-encoding gene has not been reported. In the present study, we used expressed sequence tag (EST) information to identify a cDNA from tomato that defines a new subfamily of CYP74 P-450s. We show that the protein product of the cDNA, when expressed in Escherichia coli, catalyzes the formation of CA and CnA from 9-hydroperoxides. Second, we show that this DES has spectral properties of a P-450. Finally, we demonstrate that the developmental expression of the DESencoding gene in tomato is an important determinant of the tissue specificity of divinyl ether synthesis. cDNA Sequencing-Basic molecular techniques were performed as described in Sambrook et al. (29). The clone (cLEC38I7) for tomato EST277670 (GenBank TM AW034008) was obtained from the Clemson University Genomics Institute. The cDNA insert was sequenced in its entirety on both strands. The cDNA was 1631 base pairs (bp) in length and included 18 bp upstream of the initiator AUG codon and 145 bp in the 3Ј-untranslated region (excluding 31 poly(A) residues). Thermal asymmetric interlaced polymerase chain reaction (PCR) was used to obtain additional sequence information at the 5Ј end of the gene as described previously (30,31). Briefly, 20 ng of tomato genomic DNA was used as a template for an initial PCR reaction using a gene-specific primer (GSP1: 5Ј-ACG GAT TTT TCT GAT CAC AAA GCA-3Ј) and a shorter arbitrary degenerate primer (AD3: 5Ј-(A/T)GTGNAG(A/T)AN-CANAGA)-3Ј). The resulting PCR product was used as a template for a second PCR reaction using the same AD3 primer and a second nested gene-specific primer (GSP2: 5Ј-CGT CAT AGT CGG AAG CAA AGC ATT-3Ј). The PCR product obtained from this reaction was used as template for a final PCR reaction using the AD3 primer and a third nested gene-specific primer (GSP3, 5Ј-TGT ACC ACC AAG AGT GTC AGT TTT-A-3Ј). Thermal asymmetric interlaced PCR products of ϳ1 kilobase were obtained from two independent reactions. DNA sequencing using the GSP3 primer revealed an in-frame stop codon (UAA) 27 nucleotides upstream of the first initiation AUG. Reverse transcription PCR was used to exclude the possibility that the in-frame stop codon was located in an intron that was amplified by thermal asymmetric interlaced PCR. Reverse transcription was performed with oligo(dT) primers and Superscript II reverse transcriptase (Life Technologies, Inc.). PCR was performed using a primer (5Ј-CAT CAC CTA CAA TGT TAA TA-3Ј) located upstream of the in-frame stop codon and the GSP3 primer. Direct sequencing of the reverse transcriptase PCR product confirmed the position of the stop codon relative to the initiator AUG codon. Data base searches were performed using the BLAST program (32) available at the U.S. National Center for Biotechnology. Amino acid sequence alignments were performed using the Clustal method in the Megalign program (DNAStar, Madison, WI). Expression and Purification of Recombinant CYP74D1-A PCRbased approach was used to construct the expression vector for CYP74D1 containing an N-terminal His 6 tag. Forward and reverse primers that amplify the cDNA were designed to contain BamHI and HindIII restriction sites, respectively. The sequence of the forward primer was 5Ј-CGG GAT CCC TTC CGA TTC GTG AAA TTC CA-3Ј and that of the reverse primer 5Ј-CCC AAG CTT GCA ACG TGA GCG GGC ACA CA-3Ј. PCR amplification of clone cLEC38I7 yielded a 1.45-kilobase product that was subsequently cut with BamHI and HindIII and subcloned into the same sites of expression vector pQE-30 (Qiagen Corp., Santa Clarita, CA). The resulting construct, which replaced the first nine amino acids of CYP74D1 with the sequence MRGSHHHHH-HGS, was transformed into E. coli host strain M15 for expression. A similar strategy was used for construction of the C-terminal His-tagged protein. Forward and reverse primers were designed to contain NdeI and XhoI restriction sites, respectively. The sequence of the forward primer was 5Ј-GGA ATT CCA TAT GTC TTC TTA TTC AGA GCT-3Ј and that of the reverse primer 5Ј-CCG CTC GAG TTT ACT TGC TTT AGT TAA TG-3Ј. PCR amplification of cLEC38I7 yielded a 1.45-kilobase product that was cut with NdeI and XhoI prior to subcloning into the same sites of the expression vector pET-23b (Novagen, Madison, WI). The resulting construct, which placed an additional eight amino acids (LEHHHHHH) on the C terminus of CYP74D1, was expressed in E. coli strain BL21(DE3).

Plant Materials and Growth Conditions-Tomato
His-tagged recombinant proteins were expressed in the appropriate host strains as follows. An overnight culture (5 ml) was inoculated into 100 ml of Terrific Broth medium supplemented with 200 g/ml ampicillin. Bacteria were grown at 37°C in a shaker at 250 rpm until the A 600 was 0.6 -0.8. Cultures were cooled to 25°C, and isopropyl-thio-␤-D-galactopyranoside and ␦-aminolevulinic acid were added to final concentrations of 0.05 and 0.5 mM, respectively. Induced cultures were incubated for ϳ40 h at 25°C with gentle shaking (130 rpm). Cells were collected by centrifugation and stored at Ϫ20°C until further use.
Product Analyses-Crude extracts from E. coli (M15) cells expressing CYP74D1 (N-terminal His tag form) were used as enzyme source. Extracts from cells expressing the pQE-30 vector were used as a mock control. Frozen cell paste was thawed, sonicated as described below, and centrifuged at 2,500 ϫ g for 10 min at 4°C. A portion of the supernatant corresponding to 10 mg of protein was incubated with 300 g of hydroperoxide substrate (9-HPOD or 9-HPOT) dissolved in 30 ml of 50 mM potassium phosphate buffer (pH 7.0). Reactions proceeded for 10 min at 25°C and then were stopped by acidification to pH 4.0 with 1 M citrate. Products were extracted twice with diethyl ether, dried under N 2 gas, and taken up in 0.25 ml of methanol. Extracted com-FIG. 1. Pathways for oxylipin biosynthesis. Linolenic and linoleic acids are converted to 9-and 13-hydroperoxides by LOX (R and RЈ will differ depending on the particular hydroperoxide formed). Hydroperoxide products of LOX are then metabolized by AOS, HPL, and DES to various oxylipin intermediates or end products. Other routes for hydroperoxide metabolism, including the peroxygenase and epoxyalcohol synthase pathways, are not shown. pounds were methylated by treatment with ethereal diazomethane at 0°C for 10 min, dried under N 2 gas, and taken up in 50 l of hexane. Derivatized compounds (5 l) were analyzed by GC-MS at the MSU-NIH Mass Spectrometry Facility as described previously (33). UV spectra of divinyl ether products were obtained as follows. 10 g substrate (9-HPOT or 9-HPOD) was reacted with 50 g of purified CYP74D1 in 0.2 ml of 50 mM sodium phosphate buffer for 3 min at 4°C. Ethanol (0.8 ml at Ϫ20°C) was added to the completed reaction, and precipitated protein was removed by centrifugation. A UV spectrum of the resulting supernatant was recorded with a Uvikon 933 UV/VIS spectrophotometer (Research Instruments, San Diego, CA).
Purification of Recombinant CYP74D1-Purification of the C-terminal His-tagged form of CYP74D1 was performed at 4°C except where noted. Bacterial cells expressing the construct were harvested from 200 ml of culture medium, followed by resuspension in 10 ml of buffer A (50 mM sodium phosphate, 300 mM NaCl, pH 7.0). Cells were lysed using three 30-s pulses from a probe-type sonicator (Branson Sonifier Model 450). Cell homogenates were centrifuged at 2,500 ϫ g for 10 min, and the resulting supernatant was recentrifuged at 100,000 ϫ g for 60 min. The pellet fraction containing CYP74D1 was solubilized in 2 ml of solubilization buffer (50 mM sodium phosphate, 1.5% Triton X-100R, pH 7.0) on ice for 30 min. The suspension was centrifuged at 100,000 ϫ g for 60 min, and the supernatant was diluted to 10 ml with buffer A. Following the addition of 1 ml of TALON metal affinity resin (cobaltbased IMAC, CLONTECH) pre-equilibrated with buffer A, the suspension was gently agitated at 25°C for 40 min to allow binding of CYP74D1. The resin was collected by centrifugation and washed three times at 25°C with 10 ml of buffer A containing 0.1% Triton X-100R and 5 mM imidazole (pH 7.0). The resin was applied to a column (0.7 ϫ 2.5 cm, Bio-Rad) and CYP74D1 was eluted with buffer A containing 0.1% Triton X-100R and 150 mM imidazole (pH 7.0). The reddish browncolored fractions containing the protein were pooled and concentrated in a Millipore Biomax centrifugal filter (10,000 molecular weight cut off) according to the manufacturer's instructions. Imidazole was removed by diluting the preparation ϳ10-fold with 50 mM sodium phosphate buffer (pH 7.0) containing 0.02% Triton X-100R and 5% glycerol, followed by concentration on a centrifugal filter as described above. Protein measurements were performed by the Bradford assay using bovine serum albumin as a standard. The relative purity of recombinant CYP74D1 was judged by Coomassie Brilliant Blue R-250 staining of samples analyzed on SDS-polyacrylamide gels (10% polyacrylamide).
Biochemical Analysis of CYP74D1-The hydroperoxide-metabolizing activity of recombinant CYP74D1 was measured spectrophotometrically by monitoring the rate of decrease in A 234 that results from disruption of the conjugated diene bond of the substrate (34). Kinetic assays were performed at 25°C in 1 ml of 100 mM sodium phosphate (pH 7.0) containing 30 ng of purified CYP74D1 (C-terminal His-tagged form) and varying amounts of substrate. Activity slopes obtained during the first 0.5 min of the reaction were used for calculation of kinetic parameters. Specific activity measurements were performed in a similar fashion using a substrate concentration of 50 M. Fatty acid hydroperoxide substrates were obtained from Cayman Chemical (Ann Arbor, MI). Extinction coefficients provided by the manufacturer were used for calculations. Absorbance spectra were obtained using affinity purified CYP74D1 in 1 ml of 50 mM sodium phosphate buffer (pH 7.0). Protein concentration was determined using the Bradford assay. CO treatments were performed by bubbling CO gas through the sample for 1 min, and reduction of the protein was achieved by adding a few grains of sodium dithionite.
Preparation of [1-14 C]9-HPOD-[1-14 C]9-HPOD was prepared using ripened tomato fruits as a source of 9-LOX activity, using a modification of a described previously procedure (35). Diced fruit pericarp was homogenized in 0.1 M sodium phosphate buffer (pH 7.0) containing 1 mM EDTA and 0.1% (w/v) Triton X-100R, filtered through three layers of Miracloth (Calbiochem), and centrifuged for 15 min at 10,000 ϫ g. 9-LOX activity in the supernatant was precipitated by (NH 4 ) 2 SO 4 in the 30 -60% saturated fraction. Following centrifugation for 20 min at 15,000 ϫ g, the pellet was resuspended in 0.1 M sodium phosphate buffer (pH 6.0) containing 20% (v/v) glycerol and stored at Ϫ80°C until use. [1-14 C]Linoleic acid (51 Ci mol Ϫ1 ) was purchased from PerkinElmer Life Sciences. Radiolabeled fatty acid was diluted with unlabeled linoleic acid (NuChek-Prep, Elysian, MN) to achieve a final specific activity of 1.9 Ci mol Ϫ1 . This preparation was converted to the ammonium salt and then diluted with 1% Triton X-100R to achieve a final fatty acid concentration of 10 mM. One-half ml of this solution was added to 4.5 ml of an oxygen-saturated solution containing 0.1 M sodium phosphate (pH 6.0). To this substrate was added 0.5 ml of the tomato fruit lipoxygenase fraction (12 mg of protein) described above. The reaction was allowed to proceed for 45 min at 0°C in the presence of bubbling oxygen and then stopped by acidification to pH 4.0 with 1 M citrate. Lipid products were extracted with chloroform, dried under N 2, and dissolved in a small amount of chloroform. The mixture was fractionated by TLC using a high performance silica gel plate (HP-TLC 60 F 254 , Merck) developed with diethyl ether:hexane:formic acid (70:30:1). The major radiolabeled product, [1-14 C]9-HPOD, was scraped from the TLC plate and stored in ETOH at Ϫ80°C until use.
DES Activity in Plant Extracts-A modification of a TLC assay (36) was used to monitor the transformation of [1-14 C]9-HPOD to [1-14 C]CA in cell-free extracts obtained from various tomato tissues. Briefly, extract from freshly ground roots, stems, or leaves of 18-day-old tomato seedlings was centrifuged at 10,000 ϫ g at 4°C for 2 min. A volume of supernatant containing 60 g of protein was immediately added to 0.4 ml of 40 mM sodium phosphate buffer (pH 7.0) containing 0.1% Triton X-100 and 10% glycerol (v/v). Reactions were initiated by addition of 5 g of [1-14 C]9-HPOT prepared as described above. After an incubation period of 10 min at 25°C, reactions were acidified to pH 4 and extracted with 0.4 ml of chloroform:MeOH (2:1). Lipid products obtained after evaporation of the solvent were dissolved in chloroform and separated on HP-TLC plates as described above. Labeled metabolites were visualized by autoradiography using Kodak XAR-5 film. The identity of CA among the chromatographed products was determined by GC-MS analysis of bands scraped from the TLC plate.
RNA and DNA Blot Analysis-RNA extraction and blot hybridization analysis of mRNA levels was performed as described previously (33). Full-length LeDES and LeAOS (33) cDNAs were PCR-amplified from the plasmid vector (pBlueScript) using T3 and T7 primers. Tomato genomic DNA preparations and Southern blot analysis were as described previously (33).

Identification of a Tomato cDNA Encoding a Novel CYP74
P-450 -We conducted a BLAST search (32) of the tomato EST data base for potential novel CYP74 sequences. A tentative consensus sequence, constructed from multiple overlapping ESTs (37), was identified that was similar to but clearly distinct from known CYP74 sequences in tomato and other plants. DNA sequencing of a clone (cLEC38I7) corresponding to one such EST (EST277670) revealed a 1,631-bp cDNA insert containing an open reading frame predicted to encode a 478-amino acid protein. Thermal asymmetric interlaced and reverse transcriptase PCR experiments provided additional sequence information at the 5Ј end of the transcript. DNA sequencing of this region revealed an in-frame stop codon 27 bp upstream of the first AUG, indicating that the cDNA encodes a full-length protein. A BLAST search of sequences in GenBank showed that the novel CYP74 was most similar (45% identity) to a HPL from Cucumis sativus that prefers 9-hydroperoxides to 13-hydroperoxides and is classified as CYP74C1 (38). The next strongest match (ϳ42% identity) was to members of the CYP74A subfamily (AOS). The third and final significant match (ϳ30% identity) was to HPL sequences that make up the CYP74B subfamily. According to guidelines for cytochrome P-450 nomenclature, the novel tomato CYP74 is classified as the first member (CYP74D1) of a new CYP74 subfamily designated CYP74D. 2 A comparison of the deduced CYP74D1 sequence to other known CYP74 sequences from tomato (33, 39) is shown in Fig. 2.
The sequence of CYP74D1 displayed many of the features that differentiate CYP74 enzymes from O 2 -and NADPH-requiring P-450 monooxygenases. However, some differences between CYP74D1 and other CYP74 subfamilies were noted. P-450 monooxygenases have a consensus sequence of (A/ G)GX(D/E)T(T/S) within the I-helix that forms part of the oxygen-binding pocket (40). In both bacterial and mammalian microsomal P-450, the invariant threonine residue (underlined) is thought to play a critical role in the binding and activation of oxygen (41,42). All reported CYP74 sequences have an isoleucine or valine in place of the conserved threonine, and conform to the consensus sequence GGXX(I/V)(L/F). The corresponding region of CYP74D1 has the sequence AGLNAF, which differs from all other CYP74 sequences in two positions (residues in bold type) within the consensus (Fig. 2). The hemebinding domain of CYP74D1 is characteristic of other CYP74 enzymes. The CYP74 consensus sequence for residues surrounding the cysteinyl heme ligand near the C terminus is NKQC(A/P)(G/A)K(D/N)XV and is conserved in CYP74D1. When variant positions within this consensus are considered, CYP74D1 most closely resembles cucumber HPL (CYP74C1) and two barley AOS (CYP74A) that utilize 9-and 13-hydroperoxides (38,43). The predicted sequence of CYP74D1 lacks a hydrophobic N terminus that assists in anchoring many eukaryotic P-450s to the endoplasmic reticulum (40). CYP74D1 also lacks a typical N-terminal targeting sequence that directs many CYP74 proteins, including tomato AOS (44), to the chloroplast.
CYP74D1 Is a Divinyl Ether Synthase-To investigate the catalytic function of CYP74D1, the cDNA was subcloned into vector pQE-30 for expression in E. coli. Bacterial cultures expressing the construct accumulated low levels of the recombinant protein as determined by SDS-polyacrylamide gel electrophoresis analysis of bacterial extracts (data not shown). A spectrophotometric assay was used to test extracts for their ability to metabolize fatty acid hydroperoxides (Table I). Extracts containing CYP74D1, but not those derived from control cells expressing the empty pQE30 vector, efficiently metabolized 9-HPOD and 9-HPOT. In repeated experiments using different enzyme preparations, the rate of 9-HPOD metabolism was consistently greater than that observed for 9-HPOT. Interestingly, 13-HPOT and 13-hydroperoxy linoleic acid (13-HPOD) were poor substrates for CYP74D1. This finding indicated that CYP74D1 is highly specific for the metabolism of 9-hydroperoxides. GC-MS was used to identify the methylated derivatives of metabolites produced upon incubation of CYP74D1 with 9-hydroperoxy fatty acids. With 9-HPOD as substrate, the product FIG. 2. Comparison of cDNA-deduced protein sequences of CYP74 P-450s in tomato. Sequences were aligned using the ClustalW 1.7 program. The CYP74D1 sequence is that predicted from the open reading frame of clone cLEC38I7 (EST277670). LeAOS1 (GenBank TM accession number AJ271093) and LeAOS2 (GenBank TM accession number AF230371) are members of the CYP74A subfamily. LeHPL (GenBank TM accession number AF230372) belongs to the CYP7B subfamily. Black boxes indicate amino acid residues that are identical between at least three of the four CYP74 members. Shaded boxes indicate positions that contain a conserved amino acid substitution. The hexapeptide motif (e.g. CYP74D1 residues 285-290) within the I-helix region is underlined. The ∧ symbol denotes the position of the conserved threonine found in the I-helix of P-450 monooxygenases. The CYP74 consensus sequence surrounding the cysteinyl heme ligand (*) is underlined.
UV spectroscopy was used to obtain additional structural evidence for identification of CYP74D1 products as those originally described in potato tuber (22)(23). Both CA and CnA products of CYP74D1 showed strong UV absorption, with max at 250 and 253 nm, respectively (data not shown). Mock reactions using heat-inactivated enzyme showed the characteristic spectrum ( max 234 -236 nm) of the 9-hydroperoxide substrate. These findings are in agreement with previous spectral data on CA and CnA obtained from potato tuber (22)(23)25). We conclude that CYP74D1 has DES activity that converts 9-HPOD and 9-HPOT to the divinyl ether fatty acids CA and CnA, respectively (Fig. 4). In keeping with previous nomenclature that describes the plant origin and function of CYP74 enzymes, we assigned the trivial name LeDES (L. esculentum divinyl ether synthase) to the tomato gene encoding CYP74D1.
Biochemical Properties of CYP74D1-Attempts to purify the N-terminal His-tagged form of CYP74D1 using metal affinity chromatography were unsuccessful owing to poor binding of the protein to affinity resins (data not shown). To circumvent this problem, we constructed an expression vector that places a His 6 tag on the C terminus of the protein. This form of CYP74D1 accumulated to relatively high levels in bacterial cells that were induced for expression (Fig. 5). Approximately 85% of the DES activity in the crude homogenate was recovered in the 100,000 ϫ g pellet, indicating that the protein is associated with E. coli membranes (data not shown). Solubilization of membranes with Triton X-100R and subsequent cobalt-chelate chromatography allowed purification of recombinant CYP74D1 to Ͼ95% homogeneity (Fig. 5, lanes 4 -6). The apparent molecular weight of the purified protein as determined by SDSpolyacrylamide gel electrophoresis was in good agreement with the calculated molecular weight of 55,254. Total recovery of CYP74D1 after the affinity purification step was ϳ1.2 mg/liter of cultured cells. The specific activity of the enzyme, with 9-HPOD as substrate, increased 750-fold during the purification procedure. The apparent K m of 9-HPOD and 9-HPOT was 67 and 48 M, respectively. Assuming the total protein content of CYP74D1 to be active, the estimated turnover rate (k cat ) of 9-HPOD was 890 s Ϫ1 , whereas that for 9-HPOT was 500 s Ϫ1 . These values are comparable with the substrate turnover number of AOS and HPL (17, 38, 46, 47). 13-HPOD and 13-HPOT, as well as various commercially available hydroperoxides (5-,  . 3. Mass spectra of products formed by metabolism of 9-hydroperoxides by CYP74D1. A, extracts from E. coli cells expressing CYP74D1 were incubated with 9-HPOD. Reaction products were analyzed as methyl ester derivatives by GC-MS. The major product identified, methyl colneleic acid, is shown. B, same as above, except that 9-HPOT was used as substrate. The identified product, methyl colnelenic acid, is shown. 12-, or 15-substituted) of arachidonic acid were metabolized at Ͻ 5% of the rate observed for 9-HPOD (data not shown).
The spectral properties of affinity purified CYP74D1 were typical of low spin cytochrome P-450. The UV-visible spectrum showed a Soret band at 416 nm and minor shoulders at 531 and 578 nm (Fig. 6A). Treatment of the dithionite-reduced enzyme with CO resulted in the formation of a 452-nm band, indicative of the heme-CO complex of cytochrome P-450 (Fig. 6B). This peak was highlighted in a difference spectrum between the reduced CO-heme complex and resting CYP74D1 (Fig. 6B, inset). A reproducible feature of the reduced CO spectrum was the persistence of a P420 chromophore, similar to that observed for flaxseed AOS (17). This could reflect a weak interaction of the protein with CO (17)(18) or the presence of an inactive P420 form of CYP74D1. Incomplete conversion to the P-450 form was also observed in the presence of methyl viologen, suggesting the latter possibility (data not shown).
Developmental Expression of LeDES-RNA blot analysis was used to investigate the distribution of LeDES mRNA in various organs of tomato (Fig. 7A). LeDES transcripts were most abundant in roots. A low level of LeDES mRNA was observed in stem tissue, but no accumulation was detected in flower buds, petioles, cotyledons, or leaves. This expression pattern contrasted the broad distribution of LeAOS transcripts that encode CYP74A2 (33). Previously it was reported that cell-free extracts from tomato roots but not leaves support the in vitro synthesis of CA and CnA from linoleic acid and linolenic acid, respectively (36). The results shown in Fig. 7A suggest that tissue-specific synthesis of these compounds can be accounted for by the tissue-specific expression of DES activity. To further test this hypothesis, a radio TLC assay was used to examine the ability of cell-free extracts from various tissues to catalyze the direct transformation of 9-HPOD to CA. The results showed that extracts from roots of young plants, but not extracts from stem or leaf tissue, catalyze efficient formation of CA from the hydroperoxide precursor (Fig. 7B, lanes 5-7). The identity of the labeled product as CA (lane 5) was confirmed by GC-MS (data not shown) and by its comigration with a CA standard generated with recombinant CYP74D1 (lane 4). These findings demonstrate that control of divinyl ether biosynthesis in various tomato tissues is regulated by tissue-specific expression of LeDES.
Gene Copy Number and Chromosomal Location-Tomato genomic DNA was cleaved separately with four different restriction endonucleases (BglII, DraI, EcoRI, and HindIII) and subjected to Southern blot analysis using the LeDES cDNA as a probe. The results showed a single hybridizing fragment in each of the endonuclease-digested samples (data not shown). The chromosomal location of the LeDES gene was mapped using a set of 50 introgression lines of tomato that harbor defined segments of L. pennellii DNA (48). Each line was screened for the presence of a DraI-generated RFLP that distinguishes the LeDES gene in L. pennellii from its ortholog in L. esculentum. Only one line (LA3479) displayed the L. pennellii RFLP pattern (data not shown). The introgressed DNA present in LA3479 is located on the end of the long arm of chromosome one and encompasses the region flanked by RFLP markers TG245 and TG259 on the tomato RFLP map (49). Hybridization of blots to a RFLP marker (TG369) known to map to this interval confirmed the identity of LA3479. These results show that LeDES is a single copy gene located on the distal half of the long arm of chromosome one.

DISCUSSION
Genome and EST sequencing projects have revealed plant genomes as a rich source of cytochrome P-450 genes (50). Given the diversity of substrates utilized by plant P-450 enzymes, functional assignment of P-450-encoding genes poses a significant challenge. In the case of CYP74 sequences, however, functional analysis is facilitated by two unique properties of the enzyme. First, the range of CYP74 substrates appears to be limited to fatty acid hydroperoxides. Second, the lack of a requirement for O 2 and a redox partner permits facile expression of CYP74 enzymes in E. coli. As a starting point for the present study, we identified in the EST data base a tomato cDNA predicted to encode a novel CYP74 P-450. Functional biochemical analysis of the recombinant protein showed that it catalyzes the formation of divinyl ether fatty acids CA and CnA from 9-HPOD and 9-HPOT, respectively. Although such an activity has not been reported for any P-450, a 9-hydroperoxide-specific DES (9-DES) involved in the synthesis of CA and CnA in potato tubers was described previously (22)(23). Our results suggest that the potato enzyme is a CYP74 P-450 similar to CYP74D1.
Identification of DES as a CYP74 indicates that this P-450 family plays a greater role in oxylipin metabolism than was previously realized. The majority of CYP74 sequences characterized to date encode 13-hydroperoxide-specific AOS or HPL (33,47,51). Recently, an AOS and a HPL that metabolize both 13-and 9-hydroperoxides were characterized (38,43). It is reasonable to hypothesize that other fatty acid hydroperoxide conversions, including those catalyzed by 9-AOS (52), 13-DES (24,25), and epoxy alcohol synthase (53) are mediated by CYP74 enzymes as well. The ability to identify CYP74-encoding cDNAs based solely upon DNA sequence, combined with the facile expression of these enzymes in E. coli, suggests a straightforward approach for identifying additional CYP74 members that have novel substrate specificity or catalytic activity.
DES is classified as a P-450 enzyme on the basis of both its deduced amino acid sequence and its spectral properties. Reduced heme-CO spectra recorded on the recombinant protein showed a P-450 chromophore that is the biochemical hallmark of P-450 enzymes. Identification of DES as a P-450 is consistent with the proposed mechanism of divinyl ether formation from fatty acid hydroperoxides and the chemistry of P-450-catalyzed reactions (45, 54 -56). Given the many similarities between AOS, HPL, and DES, it seems likely that hydroperoxide conversions catalyzed by these enzymes involve a similar reaction mechanism. For example, Grechkin (7) noted that all three enzymes transform hydroperoxide substrate to an epoxy allylic carbocation intermediate that is resolved differently by each enzyme to yield different oxylipin products. The epoxy allylic cation intermediate is also proposed to play a central role in the biosynthesis of oxylipins found in marine organisms (57).
The in vitro properties of recombinant CYP74D1 strongly suggest an in vivo role for the enzyme in the biosynthesis of CA and CnA (Fig. 4). By analogy to divinyl ether biosynthesis in potato (22)(23), one can propose that CYP74D1 interacts metabolically with a 9-LOX for the production of CA and CnA. In tomato, 9-LOX activity is found mainly in nonphotosynthetic tissues (root and fruit), whereas the 13-LOX pathway appears to predominate in leaves (36,58,59). This distribution may explain the observed synthesis of CA/CnA from fatty acid precursors by enzymes in tomato root extracts but not by leaf extracts (36). We found that root-specific expression of LeDES mRNA correlated with the presence of DES activity in cell-free extracts (Fig. 7). This demonstrates that the tissue-specific biosynthesis of divinyl ethers in tomato is regulated by the developmental expression of LeDES, perhaps together with 9-LOX activity. Divinyl ether synthesis may be regulated by other mechanisms as well. For example, in vitro inhibition of 9-LOX activity by CA (54) suggests that the biosynthesis of divinyl ether fatty acids or other oxylipins derived from the 9-LOX pathway may be subject to negative feedback control. Also, synthesis of CA and CnA, even in tissues that are constitutive for DES and 9-LOX activity, may require a prior inductive event for generation of fatty acid precursors. Precedence for this idea comes from recent studies showing that oxylipin synthesis in response to wounding and pathogen infection involves a phospholipase A 2 for the release of precursors from membrane lipids (60,61). Direct measurement of CA and CnA levels in healthy and afflicted tissues of tomato will be necessary to address this question. Finally, although LeDES expression was not detected in leaves of healthy tomato plants, we cannot exclude the possibility that DES and 9-LOX expression is induced in these tissues by environmental stress. This possibility is suggested by the recent observation that CA and CnA accumulate in potato and tobacco leaves challenged with pathogenic microbes (28).
Relatively little is known about the biological function of divinyl ether fatty acids. However, a recent study provided evidence that CA and CnA play an important role in resistance of potato to P. infestans, the casual agent of late blight in potato (28). A defense-related role for CA/CnA is consistent with other recent studies demonstrating the importance of the 9-LOX pathway in plant defense against fungal pathogens. Antisense expression of a 9-LOX gene was shown to suppress the incom-FIG. 6. Absorption spectra of CYP74D1. A, UV-visible spectrum of affinity purified CYP74D1 (280 g). B, spectrum of purified CYP74D1 (180 g) that was either untreated (solid line), reduced with dithionite (dotted line), or reduced with dithionite and bubbled with CO for 1 min (dashed line). The inset shows the difference spectrum obtained by subtraction of the reduced spectrum from the reduced, CO-treated spectrum. All spectra were recorded in 1 ml of 50 mM sodium phosphate buffer, pH 7.0. patible interaction between tobacco and P. parasitica, as well as to enhance susceptibility toward the compatible fungus Rhizoctonia solani (62). Another study implicated oxidized products of the 9-LOX pathway in the hypersensitive cell death induced by a fungal elicitor (63). Although the identity of specific 9-LOX-derived oxylipins that promote host defense was not determined in these studies, the work of Weber et al. (28) indicates that CA and CnA are likely components of 9-LOXmediated defense. If this is the case in tomato, the tissue specificity of DES expression suggests a role for CA/CnA in protection against pests that invade plant roots. Investigation of the biological function of divinyl ether oxylipins in plants that are genetically altered in DES expression is currently in progress.