Ivan R. Chechetkin

Unprecedented pathogen‐inducible complex oxylipins from flax – linolipins A and B

Oxylipins constitute a large family of bioregulators, biosynthesized via unsaturated fatty acid oxidation. This study reports the detection of an unprecedented family of complex oxylipins from flax leaves. Two major members of this family, compounds 1 and 2, were isolated and purified. Their structures were evaluated using NMR and MS analyses. Both compounds were identified as monogalactosyldiacylglycerol species. Compound 1 contains one α‐linolenoyl residue and one residue of (9Z,11E,1′Z,3′Z)‐12‐(1′,3′‐hexadienyloxy)‐9,11‐dodecadienoic, (ω5Z)‐etherolenic acid at positions sn‐1 and sn‐2, respectively. Compound 2 possesses (ω5Z)‐etherolenic acid residues at both position sn‐1 and position sn‐2. We suggest the trivial names linolipin A and linolipin B for compounds 1 and 2, respectively, and the collective name linolipins for this new family of complex oxylipins. The linolipin content of flax leaves increased significantly in response to pathogenesis. Thus, accumulation of esterified antimicrobial divinyl ethers may be of relevance to plant defense.

Synthesis of 3-oxalinolenic acid and β-oxidation-resistant 3-oxa-oxylipins

Abstract3-Oxalinolenic acid (3-oxa-9(Z), 12(Z), 15(Z)-octadecatrienoic acid or (6(Z), 9(Z), 12(Z)-pentadecatrienyloxy)acetic acid) was synthesized from 5(Z), 8(Z), 11(Z), 14(Z), 17(Z)-eicosapentaenoic acid by a sequence involving the C15 aldehyde 3(Z), 6(Z), 9(Z), 12(Z)-pentadecatetraenal as a key intermediate. Conversion of the aldehyde by isomerization and two steps of reduction afforded 6(Z), 9(Z), 12(Z)-pentadecatrienol, which was coupled to bromoacetate to afford after purification by HPLC >99%-pure 3-oxalinolenic acid in 10–15% overall yield. 3-Oxalinolenic acid was efficiently oxygenated by soybean lipoxygenase-1 into 3-oxa-13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid, and this hydroperoxide could be further converted chemically into 3-oxa-13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid and 3-oxa-13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The 3-oxa-hydroperoxide also served as the substrate for the plant enzymes allene oxide synthase, divinyl ether synthase, and hydroperoxide lyase to produce 3-oxa-12-oxo-10, 15(Z)-phytodienoic acid and other 3-oxa-oxylipins that were characterized by MS, 3-Oxalinolenic acid was not oxygenated by 9-lipoxygenase from tomato but was converted at a slow rate into 3-oxa-9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid by recombinant maize 9-lipoxygenase. Recombinant α-dioxygenase-1 from Arabidopsis thaliana catalyzed the conversion of 3-oxalinolenic acid into a 2-hydroperoxide, which underwent spontaneous degradation into a mixture of 6,9,12-pentadecatrienol and 6,9,12-pentadecatrienyl formate. A novel α-dioxygenase from the moss Physcomitrella patens was cloned and expressed and was found to display the same activity with 3-oxalinolenic acid as Arabidopsis thaliana α-dioxygenase-1. Lipoxygenase-generated 3-oxa-oxylipins are resistant toward β-oxidation and have the potential for displaying enhanced biological activity in situations where activity is limited by metabolic degradation.

Evidence for communality in the primary determinants of CYP74 catalysis and of structural similarities between CYP74 and classical mammalian P450 enzymes

In silico structural analysis of CYP74C3, a membrane‐associated P450 enzyme from the plant Medicago truncatula (barrel medic) with hydroperoxide lyase (HPL) specificity, showed that it had strong similarities to the structural folds of the classical microsomal P450 enzyme from rabbits (CYP2C5). It was not only the secondary structure predictions that supported the analysis but site directed mutagenesis of the substrate interacting residues was also consistent with it. This led us to develop a substrate‐binding model of CYP74C3 which predicted three amino acid residues, N285, F287, and G288 located in the putative I‐helix and distal haem pocket of CYP74C3 to be in close proximity to the preferred substrate 13‐HPOTE. These residues were judged to be in equivalent positions to those identified in SRS‐4 of CYP2C5. Significance of the residues and their relevance to the model were further assessed by site directed mutagenesis of the three residues followed by EPR spectroscopic and detailed kinetic investigations of the mutated proteins in the presence and absence of detergent. Although point mutation of the residues had no effect on the haem content of the mutated proteins, significant effects on the spin state equilibrium of the haem iron were noted. Kinetic effects of the mutations, which were investigated using three different substrates, were dramatic in nature. In the presence of detergent with the preferred substrate (13‐HPOTE), the catalytic center activities and substrate binding affinities of the mutant proteins were reduced by a factor of 8–32 and 4–12, respectively, compared with wild‐type – a two orders of magnitude reduction in catalytic efficiencies. We believe this is the first report where primary determinants of catalysis for any CYP74 enzyme, which are fully consistent with our model, have been identified. Our working model predicts that N285 is close enough to suggest that a hydrogen bond with the peroxy group of the enzyme substrate 13‐HPOTE is warranted, whereas significance of F287 may arise from a strong hydrophobic interaction between the alkyl group(s) of the substrate and the phenyl ring of F287. We believe that G288 is crucial because of its size. Any other residue with a relatively bulky side chain will hinder the access of substrate to the active site. The effects of the mutations suggests that subtle protein conformational changes in the putative substrate‐binding pocket regulate the formation of a fully active monomer‐micelle complex with low‐spin haem iron and that structural communication exists between the substrate‐ and micelle‐binding sites of CYP74C3. Conservation in CYP74 sequence alignments suggests that N285, F287, and G288 in CYP74C3 and the equivalent residues at positions in other CYP74 enzymes are likely to be critical to catalysis. To support this we show that G324 in CYP74D4 (Arabidopsis AOS), equivalent to G288 in CYP74C3, is a primary determinant of positional specificity. We suggest that the overall structure of CYP74 enzymes is likely to be very similar to those described for classical P450 monooxygenase enzymes. Proteins 2008.

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases

The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (ω6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the “pocket theory of lipoxygenase catalysis” (i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.

Isolation and structure elucidation of linolipins C and D, complex oxylipins from flax leaves

Two complex oxylipins (linolipins C and D) were isolated from the leaves of flax plants inoculated with phytopathogenic bacteria Pectobacterium atrosepticum. Their structures were elucidated based on UV, MS and NMR spectroscopic data. Both oxylipins were identified as digalactosyldiacylglycerol (DGDG) molecular species. Linolipin C contains one residue of divinyl ether (ω5Z)-etherolenic acid and one α-linolenate residue at sn-1 and sn-2 positions, respectively. Linolipin D possesses two (ω5Z)-etherolenic acid residues at both sn-1 and sn-2 positions. The rapid formation (2-30min) of linolipins C and D alongside with linolipins A and B occurred in the flax leaves upon their damage by freezing-thawing.

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 (1)H 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.

Hexadecanoid pathway in plants: Lipoxygenase dioxygenation of (7Z,10Z,13Z)-hexadecatrienoic acid

Abstract7,10,13-Hexadecatrienoic acid (16:3) is abundant in many plant species. However, its metabolism through the lipoxygenase pathway is not sufficiently understood. The goal of present work was to investigate the oxygenation of 16:3 by different plant lipoxygenases and to study the occurrence of oxygenated derivatives of 16:3 in plant seedlings. The recombinant maize 9-lipoxygenase specifically converted 16:3 into (7S)-hydroperoxide. Identification of this novel oxylipin was substantiated by data of GC-MS, LC-MS/MS, 1H-NMR, and 2D-COSY as well as by deuterium labeling from [2H6]16:3. Soybean lipoxygenase 1 produced 91% (11S)-hydroperoxide and 6% racemic 14-hydroperoxide. Recombinant soybean lipoxygenase 2 (specifically oxidizing linoleate into 13-hydroperoxide) lacked any specificity towards 16:3. Lipoxygenase 2 produced 7-, 8-, 10-, 11-, 13-, and 14-hydroperoxides of 16:3, as well as a significant amount of bis-allylic 9-hydroperoxide. Seedlings of several examined plant species possessed free hydroxy derivatives of 16:3 (HHTs), as well as their ethyl esters. Interestingly, HHTs occur not only in “16:3 plants”, but also in typical “18:3 plants” like pea and soybean seedlings.

Brassinosteroid-induced changes of lipid composition in leaves of Pisum sativum L. during senescence

HighlightsBrassinosteroids (BRs) promote the leaf senescence in concentration dependent manner.BRs‐induced senescence is accompanied by the alteration of cell lipid metabolism.BRs increase triacylglecerol and free fatty acid content while decrease galactolipids. Abstract The effect of steroid phytohormone 24‐epibrassinolide (EBR) on the composition of some lipid classes (free fatty acids, triacylglycerols and galactolipids) in detached pea leaves was studied for the first time. EBR (0.1 &mgr;M) promoted senescence and increased the content of 14:0, 16:0 and 18:1 free fatty acids as well as 18:2 and 18:3 bound to triacylglycerols in the detached leaves in contrast to mock‐treated leaves. The content of all identified fatty acids bound to galactolipids decreased in the detached leaves treated with EBR compared to that in mock‐treated leaves. These findings suggest that free fatty acids are liberated from polar lipids and then undergo esterification to neutral lipids in the detached leaves upon EBR treatment. We propose that steroid phytohormones may be involved into regulation of leaf senescence via alteration of cell lipid composition.

Brassinosteroid-induced accumulation of complex oxylipins in flax leaves

The effect of the steroid plant hormone 24-epibrassinolide on the content and composition of complex oxylipins in plants was studied for the first time, using flax as a model plant. In plants treated with the plant hormone, as well as in plants inoculated with Pectobacterium atrosepticum, the content of linolipins (galactolipids containing divinyl ether residues) increased. Hormonal pretreatment of the plants with subsequent infection resulted in a more significant accumulation of linolipins B (21-fold) and A (2.8-fold) as compared to the untreated (control) plants. The data suggest that brassinosteroids are involved in the regulation of the formation of complex oxylipins in pathogenesis of plants.

Contribution of lipoxygenase metabolism to the brassinosteroid signaling pathway.

Brassinosteroids (BSs) are phytohormones, the chemical structure of which is closest to the chemical structure of animal steroid hormones. It was shown by a number of researchers that BSs contributed to plant growth and development [1]. It was also demonstrated that BS caused an increase in the plant resistance to pathogens [2] and adverse climatic factors [3]. Receptors of BS were found to be located in plasmalemma [4]. Phosphorylation of certain proteins was demonstrated to be an early BS-induced response [5]. These findings suggest that BSs are incorporated into the signaling systems of plant cells. However, it is still unclear which types of signaling systems may incorporate BSs. It was also suggested that BSs constituted a previously unknown signaling system [2]. Because it is well known that the lipoxygenase signaling system is involved in cell response to various pathogens, mechanical injuries, elicitors, and some other primary signals [6], the goal of this work was to study the possibility of the brassinosteroid-induced activation of this signaling system. Effects of 24-epibrassinosteroid (EPB), the most active form of BS, on concentrations of oxygenated derivatives of linoleic acid (indicators of lipoxygenase activity) and on the level of phosphorylation of soluble proteins of pea leaves (it is well known that reactions of protein phosphorylation/dephosphorylation are a very important component of signaling systems) were studied. It was suggested that the analysis of lipoxygenase activity and the use of phospholipase A 2 (PLA 2 ) inhibitor provided an opportunity to elucidate the contribution of the lipoxygenase metabolism to the initial phase of the EPB-induced response of the cells. Bromphenylacyl bromide (BPB) was used as a phospholipase A 2 inhibitor decreasing the rate of release of unsaturated fatty acids (lipoxygenase substrate). The lipoxygenase activity level was assessed using the methodological approach based on the appearance of two conjugated bonds and absorption maximum at 232 nm during the reaction of formation of oxygenated derivatives of linoleic acid.