Molecular Cloning of a Divinyl Ether Synthase

Abstract

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.

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula

“…With 9-HPODE as a substrate, the recombinant StAOS3 exhibited a pH optimum of pH 6 to 6.5. To characterize the nature of the products of the enzyme reaction of StAOS3, we used [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-9-HPOTE as substrate. The reaction product, eluting at about 7 min, showed the same retention time in the HPLC-chromatograms as the α-ketol synthesized as control by unspecific 9/13-AOS from barley (Maucher et al, 2000).…”

“…To analyze if 9,10-EO(D/T), the reaction products of StAOS3, serve as substrates for the AOC from potato, we expressed StAOC in E. coli and determined the product specificity by calculating the ratio between α-ketol and cyclopentenone, eluting at about 10 min, in the radio-HPLC-chromatogram after incubation together with different AOS enzymes. When StAOC was incubated together with StAOS1 (CAD29735) and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-13-HPOTE mostly 12-OPDA was observed as product ( Figure 2b). When 13-HPODE or 9-HPO(D/T)E, respectively, were used together with StAOS1 or StAOS3 in the assay with StAOC, no AOC activity was detectable indicated by the low amount of cyclopentenones formed This is shown in Figure 2a for StAOS3 together [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-9-HPOTE.…”

“…Incubation of LA or LnA with potato tuber or tomato root protein extracts led to the formation of fatty acid 9-hydroperoxides which are further metabolized to divinyl ethers, such as colneleic (CA) or colnelenic acid (CnA), respectively [2]. The DES responsible for the CA and CnA formation in potato and tomato have been cloned and characterized [4,5]. The analysis of their sequences led to classification of the enzymes within the group of cytochrome P450 enzymes, specifically in the CYP74D subfamily.…”

Identification of a Jasmonate-regulated Allene Oxide Synthase That Metabolizes 9-Hydroperoxides of Linoleic and Linolenic Acids

“…RNA Blot Analysis and Gene Mapping-RNA and DNA blot analyses were performed as previously described (13,23). Total RNA was extracted from soil-grown plants or from seeds (4 days after imbibition) that were germinated on water-saturated filter paper.…”

“…The cDNA insert, which was sequenced in its entirety on both strands, was 1773 base pairs (bp) in length, and included 16-bp upstream of the initiator AUG codon and 281 bp in the 3Ј-untranslated region (excluding poly(A) residues). Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was used to obtain additional sequence information at the 5Ј-end of the gene as previously described (23). 20 ng of tomato genomic DNA was used as a template for an initial PCR reaction using a gene-specific primer (GSP1: 5Ј-TGG-AGT-TGT-AGA-ACG-CGT-TGT-ATA-GCT-TC-3Ј) and a shorter arbitrary degenerate primer (AD1: 5Ј-(A/C/G/T)TCGA(C/G)T(A/T)T(C/ G)G(A/T)GTT-3Ј).…”