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 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.     

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.     

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.     

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.     

S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures

An S-adenosyl-L-methionine:caffeoyl-CoA 3-O-methyltransferase was purified 82-fold from elicitor-induced parsley cell suspension cultures by ammonium sulfate fractionation, anionic exchange and hydrophobic interaction chromatographies, and chromatofocusing. The enzyme has an apparent pI of 5.7 and a molecular weight of approx 48,000 determined by gel filtration chromatography. Maximal activity was observed at pH 7.5 in 50 mM phosphate or Tris-HCl buffers and the additional presence of 0.5 M NaCl. The methyltransferase activity was dependent on Mg2+, whereas EDTA, Mn2+, and Ca2+ inhibited the reaction. The partially purified enzyme efficiently catalyzed the methylation of caffeoyl-CoA, but also accepted with low affinity various other caffeic esters as substrates. Dark-grown parsley cells contained considerable methyltransferase activity which was nevertheless increased approx threefold within 12 h following the addition of a crude fungal elicitor to the cell suspensions. We propose that the O-methyltransferase activity is an important component in the rapid resistance response of the cells, which depends on the formation of cell wall-bound ferulic polymers.     

Purification and inactivation by substrate of an allene oxide synthase (CYP74) from corn (Zea mays L.) seeds

The allene oxide synthase (AOS) was purified from corn (Zea mays) seeds to homogeneity and characterized partially. The corn AOS was a hemoprotein cytochrome P450 with a molecular weight and pI of 53,000 and 6.0, respectively. The corn AOS was found to be irreversibly inactivated by a substrate, 13-hydroperoxyoctadienoic acid. The rate of the enzyme inactivation was higher at low pHs.     

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.     

Design and synthesis of novel imidazole derivatives as potent inhibitors of allene oxide synthase(CYP74)

Allene oxide synthase (AOS) is a key enzyme in the oxylipin pathway in plants leading to jasmonic acid and other jasmonates (JAs), important signal mediators of defense signal networks in plants. AOS uses hydroperoxylinolenic acid as an oxygen donor as well as the substrate, thus the biochemical conversion of 13(S)-hydroperoxylinolenic acid to allene oxide can proceed in the absence of oxygen and NADPH. We have designed the synthesized of a series of novel imidazole derivatives and tested them in a bioassay as AOS inhibitors using a purified recombinant AOS enzyme isolated from Arabidopsis and expressed in E. coli. Among the derivatives prepared, heptyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]octanoate (k) was found to be the most potent inhibitor, with an IC₅₀ of 10±5 nM, which is 250,000-fold and 1,000,000-fold more potent than the known AOS inhibitors, acetylsalicyclic acid (2.5 mM) and ketoconazole (10 mM), respectively.     

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.     

Induction of freezing tolerance in potato (Solanum commersonü) suspension cultured cells

The induction of freezing tolerance by abscisic acid (ABA) or cold treatment in suspension cultured cells of Solanum commersonii was studied. Both ABA (50–100 μ M ) at 23°C and low temperature (4°C) increased freezing tolerance in cultured Solanum commersonii cells from a LT₅₀ (freezing temperature at which 50% cells were killed) of —5°C (control) to —11.5°C in 2 days. Cold-induced freezing tolerance reached its maximum at 2 days and remained constant throughout the cold acclimation period of 11 days. The freezing tolerance induced by ABA, however, showed a rapid decline 2 to 5 days after initiation of ABA treatments. Addition of ABA (100 μ M ) to the culture medium at the inception of low temperature treatment did not enhance freezing tolerance of the cells beyond the level attainable by either treatment singly. Poly(A⁺)-RNA was isolated from the respective treatments, translated in a rabbit reticulocyte lysate cell free system, and the translation products were resolved by two dimensional polyacrylamide gel electrophoresis (ID-PAGE). Analysis of the in vitro translated products revealed changes in the abundance of approximately 26 products (encoding for polypeptides with M, of 14 to 69 kDa and pl of 4.90 to 6.60) in ABA-treated cells 12 h after treatment, and 20 (encoding for polypeptides with M_r of 12 to 69 kDa, with pl of 4.80 to 6.42) in cells exposed to 4°C for 12 h. There were only 5 novel translation products observed when the ABA-treated cells reached the highest level of freezing tolerance (2 days after the initiation of ABA treatment). Changes in translatable RNA populations during the induction of freezing tolerance in cells treated with either ABA or low temperature are discussed.     

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.     

Specificity of starch synthase isoforms from potato.

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.     

Epoxyalcohol synthase of Ectocarpus siliculosus. First CYP74-related enzyme of oxylipin biosynthesis in brown algae

Enzymes of CYP74 family play the central role in the biosynthesis of physiologically important oxylipins in land plants. Although a broad diversity of oxylipins is known in the algae, no CYP74s or related enzymes have been detected in brown algae yet. Cloning of the first CYP74-related gene CYP5164B1 of brown alga Ectocarpus siliculosus is reported in present work. The recombinant protein was incubated with several fatty acid hydroperoxides. Linoleic acid 9-hydroperoxide (9-HPOD) was the preferred substrate, while linoleate 13-hydroperoxide (13-HPOD) was less efficient. α-Linolenic acid 9- and 13-hydroperoxides, as well as eicosapentaenoic acid 15-hydroperoxide were inefficient substrates. Both 9-HPOD and 13-HPOD were converted into epoxyalcohols. For instance, 9-HPOD was turned primarily into (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acid. Both epoxide and hydroxyl oxygen atoms of the epoxyalcohol were incorporated mostly from [¹⁸O₂]9-HPOD. Thus, the enzyme exhibits the activity of epoxyalcohol synthase (EsEAS). The results show that the EsEAS isomerizes the hydroperoxides into epoxyalcohols via epoxyallylic radical, a common intermediate of different CYP74s and related enzymes. EsEAS can be considered as an archaic prototype of CYP74 family enzymes.     

Expression of a vacuolar protein (VP24) in anthocyanin-producing cells of sweet potato in suspension culture.

VP24, an abundant protein of 24 kD, was found to accumulate in the anthocyanin-containing vacuoles of cells of sweet potato (Ipomoea batatas) in suspension culture. Light-induced expression of VP24 was analyzed by immunoblotting in three different cell lines that produced anthocyanins at different rates. The expression of VP24 was closely correlated with the accumulation of anthocyanin in these cell lines. Immunocytochemical detection of VP24 with specific antibodies on thin sections showed that VP24 was localized in the intravacuolar pigmented globules (cyanoplasts) in the anthocyanin-containing vacuoles and not in the tonoplast. No VP24 immunogold labeling was detected in the vacuoles of the cell line that does not produce anthocyanin. We suggest that VP24 may be involved in the formation of the cyanoplast via an interaction with anthocyanin, and that it may play an important role in the trapping in vacuoles of large amounts of anthocyanins that have been transported into these vacuoles.     

Geometrical configuration of 12,13-epoxy-9,11-octadecadienoic acid, a product of the reaction catalyzed by flaxseed allene oxide synthase (CYP74A)

The geometrical configuration of a short-living allene oxide reaction product that arises under the catalysis by flaxseed allene oxide synthase (CYP74A) was studied by NMR spectroscopy. The structure of (9Z,11E)-12,13-epoxyoctadeca-9,11-dienoic acid was established for it from the results of the nuclear Overhauser effect. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Oleosomes (Spherosomes) from Daucus carota suspension culture cells

Isolated oleosomes from Daucus carota L. cells are lipid droplets consisting mainly of triacylglycerols (>97%) and very little protein (1–2%). The boundary between the lipid phase and the cytosol, which is visible on electron micrographs, is not built up by a true phospholipid-containing unit or half unit membrane. Enzymatic activities of lipid metabolism were not found to be associated with oleosomes with the exception of very low (contaminating) acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) and relatively high acyl-CoA hydrolase (EC 3.1.2.2) activities. The triacylglycerols exhibited a half life time of about 70 h, which is below the generation time of the cells (80–90 h). The fatty acid pattern of triacylglycerols was very similar to that of polar cellular membrane lipids.