A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

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.     

Detection of unprecedented allene oxide synthase member of CYP74B subfamily: CYP74B33 of carrot (Daucus carota)

Enzymes of CYP74 family widespread in higher plants control the metabolism of fatty acid hydroperoxides to numerous bioactive oxylipins. Hydroperoxide lyases (HPLs, synonym: hemiacetal synthases) of CYP74B subfamily belong to the most common CYP74 enzymes. HPLs isomerize the hydroperoxides to the short-lived hemiacetals, which are spontaneously decomposed to aldehydes and aldoacids. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) possessed the 13-HPL activity. Present work reports the cloning of the expressed CYP74B33 gene of carrot (Daucus carota L.) and studies of catalytic properties of the recombinant CYP74B33 protein. In contrast to all CYP74B proteins studied yet, CYP74B33 behaved differently in few respects. Firstly, the preferred substrates of CYP74B33 are 9-hydroperoxides. Secondly and most importantly, CYP74B33 exhibits the 9-allene oxide synthase (AOS) activity. For example, the 9(S)-hydroperoxide of linoleic acid (9-HPOD) underwent the conversion to α-ketol via the short-lived allene oxide. Uncommonly, the 9-HPOD conversion affords a minority of cis-10-oxo-11-phytoenoic acid, which is also produced by CYP74C but not the CYP74A AOSs. The similar product patterns were observed upon the incubations of CYP74B33 with 9(S)-hydroperoxide of α-linolenic acid. The enzyme possessed a mixed HPL, AOS, and the epoxyalcohol synthase activity toward the 13-hydroperoxides, but the total activity was much lower than toward 9-hydroperoxides. Thus, the obtained results show that CYP74B33 is an unprecedented 9-AOS within the CYP74B subfamily.     

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.     

Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3

Nonclassical P450s of CYP74 family control the secondary conversions of fatty acid hydroperoxides to bioactive oxylipins in plants. At least ten genes attributed to four novel CYP74 subfamilies have been revealed by the recent sequencing of the spikemoss Selaginella moellendorffii Hieron genome. Two of these genes CYP74M1 and CYP74M3 have been cloned in the present study. Both recombinant proteins CYP74M1 and CYP74M3 were active towards the 13(S)-hydroperoxides of α-linolenic and linoleic acids (13-HPOT and 13-HPOD, respectively) and exhibited the activity of divinyl ether synthase (DES). Products were analyzed by gas chromatography–mass spectrometry. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the ¹H NMR, 2D-COSY, HSQC and HMBC. CYP74M1 (SmDES1) specifically converted 13-HPOT to (11Z)-etherolenic acid and 13-HPOD to (11Z)-etheroleic acid. CYP74M3 (SmDES2) turned 13-HPOT and 13-HPOD mainly to etherolenic and etheroleic acids, respectively. CYP74M1 and CYP74M3 are the first DESs detected in non-flowering plants. The obtained results demonstrate the existence of the sophisticated oxylipin biosynthetic machinery in the oldest taxa of vascular plants.     

Formation of oxylipins by CYP74 enzymes

Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes. Products are hydroperoxy polyunsaturated fatty acids and metabolites derived there from collectively named oxylipins. They may either originate from chemical oxidation or are synthesized by the action of various enzymes, such as lipoxygenases. Cloning of many lipoxygenases and other key enzymes metabolizing oxylipins revealed new insights on oxylipin functions, new reactions and the first hints on enzyme mechanisms. These aspects are reviewed with respect to metabolism of fatty acid hydroperoxides by an atypical P450 subfamily: the CYP74. Up to now this protein family contains three different enzyme activities: (i) allene oxide synthase leading to the formation of unstable allene oxides which react to ketol and cyclopentenone fatty acids, (ii) hydroperoxide lyase producing hemiacetals decomposing to aldehydes and ω-oxo fatty acids and (iii) divinyl ether synthase which forms divinyl ethers. Signalling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among their numerous products.     

Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of fatty acid hydroperoxides into hemiacetals

The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.     

Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.)

Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries.     

Identification and functional analyses of two cDNAs that encode fatty acid 9-/13-hydroperoxide lyase (CYP74C) in rice

Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.     

Identification of an allene oxide synthase (CYP74C) that leads to formation of alpha-ketols from 9-hydroperoxides of linoleic and linolenic acid in below-ground organs of potato

Allene oxide synthase (AOS) enzymes are members of the cytochrome P450 enzyme family, sub-family CYP74. Here we describe the isolation of three cDNAs encoding AOS from potato (StAOS1-3). Based on sequence comparisons, they represent members of either the CYP74A (StAOS1 and 2) or the CYP74C (StAOS3) sub-families. StAOS3 is distinguished from the other two AOS isoforms in potato by its high substrate specificity for 9-hydroperoxides of linoleic and linolenic acid, compared with 13-hydroperoxides, which are only poor substrates. The highest activity was shown with (9S,10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) as a substrate. This hydroperoxide was metabolized in vitro to alpha- and gamma-ketols as well as to the cyclopentenone compound 10-oxo-11-phytoenoic acid. They represent hydrolysis products of the initial StAOS3 product 9,10-epoxyoctadecadienoic acid, an unstable allene oxide. By RNA gel hybridization blot analysis, StAOS3 was shown to be expressed in sprouting eyes, stolons, tubers and roots, but not in leaves. StAOS3 protein was found in all organs tested, but mainly in stems, stolons, sprouting eyes and tubers. As in vivo reaction products, the alpha-ketols derived from 9-hydroperoxides of linoleic and linolenic acid were only found in roots, tubers and sprouting eyes. Immunolocalization showed that StAOS3 was associated with amyloplasts and leucoplasts.     

Hydroperoxide lyase cascade in pea seedlings: Non-volatile oxylipins and their age and stress dependent alterations

The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography-mass spectrometry after invitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic, (10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that: (1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings; (2) the HPL products may have the regulatory role both in growth control and adaptation.     

Hidden stereospecificity in the biosynthesis of divinyl ether fatty acids

Incubations of [8(R)-2H]9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid, [14(R)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid and [14(S)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid were performed with preparations of plant tissues containing divinyl ether synthases. In agreement with previous studies, generation of colneleic acid from the 8(R)-deuterated 9(S)-hydroperoxide was accompanied by loss of most of the deuterium label (retention, 8%), however, the opposite result (98% retention) was observed in the generation of 8(Z)-colneleic acid from the same hydroperoxide. Formation of etheroleic acid and 11(Z)-etheroleic acid from the 14(R)-deuterated 13(S)-hydroperoxide was accompanied by loss of most of the deuterium (retention, 7-8%), and, as expected, biosynthesis of these divinyl ethers from the corresponding 14(S)-deuterated hydroperoxide was accompanied by retention of deuterium (retention, 94-98%). Biosynthesis of omega5(Z)-etheroleic acid from the 14(R)- and 14(S)-deuterated 13(S)-hydroperoxides showed the opposite results, i.e. 98% retention and 4% retention, respectively. The experiments demonstrated that biosynthesis of divinyl ether fatty acids from linoleic acid 9- and 13-hydroperoxides takes place by a mechanism that involves stereospecific abstraction of one of the two hydrogen atoms alpha to the hydroperoxide carbon. Furthermore, a consistent relationship between the absolute configuration of the hydrogen atom eliminated (R or S) and the configuration of the introduced vinyl ether double bond (E or Z) emerged from these results. Thus, irrespective of which hydroperoxide regioisomer served as the substrate, divinyl ether synthases abstracting the pro-R hydrogen generated divinyl ethers having an E vinyl ether double bond, whereas enzymes abstracting the pro-S hydrogen produced divinyl ethers having a Z vinyl ether double bond.     

Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots

Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1′E)-12-(1′-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, ¹H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1′E,3′Z)-12-(1′,3′-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.     

CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in <Emphasis Type="Italic">Eschscholzia californica</Emphasis>

Eschscholzia californica produces various types of isoquinoline alkaloids. The structural diversity of these chemicals is often due to cytochrome P450 (P450) activities. Members of the CYP719A subfamily, which are found only in isoquinoline alkaloid-producing plant species, catalyze methylenedioxy bridge-forming reactions. In this study, we isolated four kinds of CYP719A genes from E. californica to characterize their functions. These four cDNAs encoded amino acid sequences that were highly homologous to Coptis japonica CYP719A1 and E. californica CYP719A2 and CYP719A3, which suggested that these gene products may be involved in isoquinoline alkaloid biosynthesis in E. californica, especially in methylenedioxy bridge-forming reactions. Expression analysis of these genes showed that two genes (CYP719A9 and CYP719A11) were preferentially expressed in plant leaf, where pavine-type alkaloids accumulate, whereas the other two showed higher expression in root than in other tissues. They were suggested to have distinct physiological functions in isoquinoline alkaloid biosynthesis. Enzyme assay analysis using recombinant proteins expressed in yeast showed that CYP719A5 had cheilanthifoline synthase activity, which was expected based on the similarity of its primary structure to that of Argemone mexicana cheilanthifoline synthase (deposited at DDBJ/GenBanktrade mark/EMBL). In addition, enzyme assay analysis of recombinant CYP719A9 suggested that it has methylenedioxy bridge-forming activity toward (R,S)-reticuline. CYP719A9 might be involved in the biosynthesis of pavine- and/or simple benzylisoquinoline-type alkaloids, which have a methylenedioxy bridge in an isoquinoline ring, in E. californica leaf.