C6-aldehyde formation by fatty acid hydroperoxide lyase in the brown alga Laminaria angustata

Some marine algae can form volatile aldehydes such as n-hexanal, hexenals, and nonenals. In higher plants it is well established that these short-chain aldehydes are formed from C18 fatty acids via actions of lipoxygenase and fatty acid hydroperoxide lyase, however, the biosynthetic pathway in marine algae has not been fully established yet. A brown alga, Laminaria angustata, forms relatively higher amounts of C6- and C9-aldehydes. When linoleic acid was added to a homogenate prepared from the fronds of this algae, formation of n-hexanal was observed. When glutathione peroxidase was added to the reaction mixture concomitant with glutathione, the formation of n-hexanal from linoleic acid was inhibited, and oxygenated fatty acids accumulated. By chemical analyses one of the major oxygenated fatty acids was shown to be (S)-13-hydroxy-(Z, E)-9, 11-octadecadienoic acid. Therefore, it is assumed that n-hexanal is formed from linoleic acid via a sequential action of lipoxygenase and fatty acid hydroperoxide lyase (HPL), by an almost similar pathway as the counterpart found in higher plants HPL partially purified from the fronds has a rather strict substrate specificity, and only 13-hydroperoxide of linoleic acid, and 15-hydroperoxide of arachidonic acid are the essentially suitable substrates for the enzyme. By surveying various species of marine algae including Phaeophyta, Rhodophyta and Chlorophyta it was shown that almost all the marine algae have HPL activity. Thus, a wide distribution of the enzyme is expected.     

Suicidal inactivation of the rabbit 15-lipoxygenase by 15S-HpETE is paralleled by covalent modification of active site peptides

Lipoxygenases (LOXs) are multifunctional enzymes that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives; they also convert hydroperoxy fatty acids to epoxy leukotrienes and other secondary products. LOXs undergo suicidal inactivation but the mechanism of this process is still unclear. We investigated the mechanism of suicidal inactivation of the rabbit 15-lipoxygenase by [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid (15-HpETE) and observed covalent modification of the enzyme protein. In contrast, nonlipoxygenase proteins (bovine serum albumin and human gamma-globulin) were not significantly modified. Under the conditions of complete enzyme inactivation we found that 1.3 +/- 0.2 moles (n = 10) of inactivator were bound per mole lipoxygenase, and this value did depend neither on the enzyme/inactivator ratio nor on the duration of the inactivation period. Covalent modification required active enzyme protein and proceeded to a similar extent under aerobic and anaerobic conditions. In contrast, [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE), which is no substrate for epoxy-leukotriene formation, did not inactivate the enzyme and protein labeling was minimal. Separation of proteolytic cleavage peptides (Lys-C endoproteinase digestion) by tricine SDS-PAGE and isoelectric focusing in connection with N-terminal amino acid sequencing revealed covalent modification of several active site peptides. These data suggest that 15-lipoxygenase-catalyzed conversion of (15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid to 14,15-epoxy-leukotriene leads to the formation of reactive intermediate(s), which are covalently linked to the active site. Therefore, this protein modification contributes to suicidal inactivation.     

Fatty acid hydroperoxide lyase in tomato fruits: cloning and properties of a recombinant enzyme expressed in Escherichia coli

Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a lambda max at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.     

Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C(18) fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.     

Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.     

Detection of an enol intermediate in the hydroperoxide lyase chain cleavage reaction

Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-(14)C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0 degrees C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography-mass spectrometry and radio-high-performance liquid chromatography revealed a single predominant (14)C-labelled compound, identified by its (1)H-nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)-12-oxo-9-dodecenoic acid. The data obtained demonstrate that formation of (9Z)-12-oxo-9-dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis.     

Activity of soybean lipoxygenase isoforms against esterified fatty acids indicates functional specificity

In soybean (Glycine max L.) vegetative tissue at least five lipoxygenase isozymes are present. Four of these proteins have been localized to the paraveinal mesophyll, a layer of cells that is thought to function in assimilate partitioning. In order to determine the role of the lipoxygenase isozymes within the soybean plant, the leaf lipoxygenases were cloned into bacterial expression vectors and expressed in Escherichia coil. The recombinant lipoxygenases were then characterized as to substrate preference, pH profiles for the most common plant lipoxygenase substrates, linoleic acid, and alpha-linolenic acid, and the reaction products with the substrates linoleic acid, alpha-linolenic acid, arachidonic acid, gamma-linolenic acid, and the triacylglycerol trilinolein. All five enzymes were shown to be (13S)-lipoxygenases against linoleic acid. The results of these assays also indicate that two of these isozymes are highly active against esterified fatty acid groups, such as those found in triacylglycerols. Lipid analysis of leaves from plants subjected to sink limitation conditions indicates that the soybean leaf lipoxygenases are active in vivo against both free fatty acids and esterified lipids, and that the quantities of lipoxygenase products found in leaf tissue show a positive correlation with the level of lipoxygenase in the leaf. Implications for the putative role of these enzymes in the paraveinal mesophyll are discussed.     

Activity of hydroperoxide lyase under aqueous and micro-aqueous conditions

Hydroperoxide lyases (HPL E.C. 4.1.2.) are part of the lipoxygenase pathway in plants and catalyze the conversion of fatty acid hydroperoxides into oxo acids and short chain aldehydes. These aldehydes have desirable properties for the food and agricultural industry. HPL activity can be modulated by salts and surfactants, but the mechanisms governing the modulation are not fully understood. Recombinant HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. To investigate solubility issues of the two different substrates, an aqueous and a two-phase micro-aqueous reaction medium was used. The highest HPL activity (8.7 μmol min⁻¹ mg⁻¹) was achieved under aqueous conditions with high salt (1.5 M) and low surfactant (0%, v/v) concentrations and LA-OOH as a substrate. Maximal activity (2.4 μmol min⁻¹ mg⁻¹) under micro-aqueous conditions was achieved with high salt (1.5 M) and high surfactant (0.01%, v/v) concentrations and LS-OOH as a substrate. A significant interaction between salt and surfactant as well as salt and substrate could be identified and a hypothesis for the interaction phenomena is presented.     

Oxylipin metabolism in the red alga Gracilariopsis lemaneiformis: mechanism of formation of vicinal dihydroxy fatty acids

Conversion of arachidonic acid into the vicinal diol fatty acid 12R,13S-dihydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid using an acetone powder of the marine red alga, Gracilariopsis lemaneiformis, occurred via intermediate formation of 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid. Incubations of the linoleic acid-derived 13S- and 13R-hydroperoxy-9Z,11E-octadecadienoic acids led to the formation of 13R,14S-dihydroxy-9Z,11E-octadecadienoic acid and 13S,14S-dihydroxy-9Z,11E-octadecadienoic acid, respectively, whereas incubation of 9S-hydroperoxy-10E,12Z-octadecadienoic acid resulted in the formation of 8S,9R-dihydroxy-10E,12Z-octadecadienoic acid. Experiments with 18O2-labeled 13S-hydroperoxyoctadecadienoic acid demonstrated that the oxygens of the two hydroxyl groups of 13R,14S-dihydroxy-9Z,11E-octadecadienoic acid originated in the hydroperoxy group of the substrate. Furthermore, experiments with mixtures of unlabeled and 18O2-labeled 13S-hydroperoxyoctadecadienoic acid showed that conversion into 13R,14S-dihydroxyoctadecadienoic acid occurred by a reaction involving an intramolecular hydroxylation at C-14 by the distal hydroperoxide oxygen. The existence of a hydroperoxide isomerase in G. lemaneiformis which catalyzes the conversion of fatty acid hydroperoxides into vicinal diol fatty acids is postulated.     

The homolytic and heterolytic fatty acid hydroperoxide lyase-like activities of hematin

Pentenols and pentene dimers are biosynthetized in plants by homolytic fatty acid hydroperoxide lyase (HPL) or HPL-like enzymes. It has been found that these compounds can modify the flavor of olive oil. Reactions between hematin and 13-hydroperoxyoctadecatrienoic acid resulted in the formation of the same compounds via a free radical reaction in which an alkoxyl radical derived from linolenic acid hydroperoxide undergoes a beta-scission. (Z)-3-Hexenal has also been detected as a minor product of the reaction. It is bioconversed from the same substrate in plants by heterolytic HPL. Thanks to the redox cycle of its central iron, hematin has both homolytic and heterolytic HPL-like activities.     

A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.     

Substrate specificity of acyl-Delta(6)-desaturases from Continental versus Macaronesian Echium species

Echium (Boraginaceae) species from the Macaronesian islands exhibit an unusually high level of gamma-linolenic acid (18:3n-6; GLA) and relatively low content of octadecatetraenoic acid (18:4n-3; OTA) in the seed, while the amounts of both fatty acids in their Continental (European) relatives are rather similar. We have tested the hypothesis of whether a different specificity of the acyl-Delta(6)-desaturases (D6DES) towards their respective usual substrates, linoleic acid (18:2n-6; LA) for GLA and alpha-linolenic acid (18:3n-3; ALA) for OTA, was partly responsible for this composition pattern. To this aim we have expressed in yeast the coding sequences of the D6DES genes for the Continental species Echium sabulicola, and the Macaronesian Echium gentianoides. When the yeast cultures are supplemented with the two fatty acid substrates (LA and ALA), a similar utilization of both compounds was found for the D6DES of E. sabulicola, while a preference for LA over ALA was observed for the enzyme of E. gentianoides. This substrate preference must contribute to the increased accumulation of GLA in the seeds of the Macaronesian Echium species. Comparison among the amino acid sequences of these desaturases and other related enzymes, allowed us the discussion about the possible involvement of some specific positions in the determination of substrate specificity.     

Molecular cloning and expression of Arabidopsis fatty acid hydroperoxide lyase.

Fatty acid hydroperoxide lyase (HPOL), an enzyme of the octadecanoid pathway that forms carbon-6 aldehydes such as n-hexanal or (Z)-3-hexenal, was cloned from Arabidopsis thaliana as a full-length cDNA. The HPOL activity obtained by expressing the cDNA in Escherichia coli formed n-hexanal from linoleic acid 13-hydroperoxide, whereas linoleic acid 9-hydroperoxide was not a substrate for the enzyme. The HPOL mRNA is expressed at low level in leaves; however, its accumulation can be found in the inflorescence. Wounding or methyl jasmonate treatments increase the mRNA level in leaves. These results indicate that the HPOL gene is up-regulated in leaves in response to wounding and that the enzyme may be an active component of the octadecanoid defense response.     

Analysis of a specific oxygenation reaction of soybean lipoxygenase-1 with fatty acids esterified in phospholipids

Soybean lipoxygenase was reacted with phosphatidylcholine (at pH 9, with 10 mM deoxycholate), and the oxygenation products were analyzed by high-pressure liquid chromatography, UV, gas chromatography-mass spectrometry (GC-MS), and NMR. The structures of the intact glycerolipid products were established by GC-MS of diglycerides recovered by phospholipase C hydrolysis and by proton NMR of the intact phosphatidylcholine. These analyses, together with analyses of the transesterified fatty acids, indicated that arachidonyl and linoleoyl moieties in the phosphatidylcholine were converted exclusively to the 15(S)-hydroperoxy-5(Z),8(Z),11(Z),13(E)-eicosatetraenoate and 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate analogues, respectively. Control experiments proved that the intact phospholipid (and not hydrolyzed/reesterified fatty acid) was the true substrate of the oxygenation reaction. Phosphatidylethanolamine and phosphatidylinositol lipids were also substrates for specific oxygenation by the soybean lipoxygenase. The results provide concrete evidence that fatty acids esterified in phospholipid can be subject to highly specific oxygenation by a lipoxygenase enzyme.     

Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 1: NMR spectral characterization

Methyl esters of gamma-linolenic acid, alpha-linolenic acid and stearidonic acid were epoxidised using m-chloroperbenzoic acid to achieve nine cis-monoepoxy-C18 fatty acid methyl esters (FAMEs), including novel methyl cis-monoepoxy derivatives of stearidonic acid and a cis-6,7-epoxy derivative of gamma-linolenic acid. These nine monoepoxy FAMEs were purified by normal-phase HPLC, identified by LC-MS, 1H and 13C NMR, and characterized by mass spectrometry and NMR spectroscopy. This study is focused on structural characterization of these C18 monoepoxy FAMEs using techniques in NMR spectroscopy including 1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear correlation (HETCOR). The proton and carbon NMR chemical shifts of the epoxide, the double bonds, and the interrupted methylenes are assigned. Also discussed is an interpretation of the 1H and 13C NMR spectra of these monoepoxides including the changes in the 13C resonance of the olefinic carbons on the neighboring double bonds resulting from epoxide formation.     

高山被孢霉ATCC16266△^6—脂肪酸脱氢酶基因在酿酒酵母中的表达

△^6－脂肪酸脱氢酶基因是形成γ－亚麻酸的关键酶。从含有高山被孢霉△^6－脂肪酸脱氢酶基因的重组质粒pT-MACL6中,酶切出1.4kb的目的片段,亚克隆到大肠杆菌和酿酒酵母的穿梭表达载体pYES2.0,在大肠杆菌中筛选到含有目的基因的重组质粒pYMAD6,用醋酸昔方法转化到酿洒酵母的缺陷型菌株INCSc1中,在SC－Ura合成培养基中,选择得到酿酒酵母工程株YMAD6。在合适的培养基及培养条件下,加入外源底物亚油酸,经半乳糖诱导后,收集菌体。通过GC－MS对酵母工程株进行脂肪酸色谱分析,结果表明,产生了31.6％的γ－亚麻酸,边是迄今为止,国内外△^6－脂肪酸脱氢酶基因在酿酒酵母中表达量最高的报道。     

The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

Potato tubers exhibit both homolytic and heterolytic hydroperoxide fatty acid-cleaving activities

The action of a crude potato-tuber extract on 9- and 13-hydroperoxides of linoleic and linolenic acids was investigated. HPLC analysis revealed that 50% of the 9-hydroperoxide isomers and almost all the 13-hydroperoxide isomers were rapidly enzymically metabolized. No degradation of fatty acid hydroperoxides was observed with a thermally denatured enzymic extract. GC-MS identification of the volatiles formed by the reaction revealed that no volatiles were detected from the 9-hydroperoxide isomers, whereas 13-hydroperoxide of linolenic acid was cleaved into (Z)-3-hexenal, pentenols or dimers of pentene.     

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.     

A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis. Biosynthesis of (8R)-hydroxylinoleic acid and (7S,8S)-dihydroxylinoleic acid from (8R)-hydroperoxylinoleic acid.

The fungus Gaeumannomyces graminis metabolized linoleic acid extensively to (8R)-hydroperoxylinoleic acid, (8R)-hydroxylinoleic acid, and threo-(7S,8S)-dihydroxylinoleic acid. When G. graminis was incubated with linoleic acid under an atmosphere of oxygen-18, the isotope was incorporated into (8R)-hydroxylinoleic acid and 7,8-dihydroxylinoleic acid. The two hydroxyls of the latter contained either two oxygen-18 or two oxygen-16 atoms, whereas a molecular species that contained both oxygen isotopes was formed in negligible amounts. Glutathione peroxidase inhibited the biosynthesis of 7,8-dihydroxylinoleic acid. These findings demonstrated that the diol was formed from (8R)-hydroperoxylinoleic acid by intramolecular hydroxylation at carbon 7, catalyzed by a hydroperoxide isomerase. The (8R)-dioxygenase appeared to metabolize substrates with a saturated carboxylic side chain and a 9Z-double bond. G. graminis also formed omega 2- and omega 3-hydroxy metabolites of the fatty acids. In addition, linoleic acid was converted to small amounts of nearly (65% R) racemic 10-hydroxy-8,12-octadecadienoic acid by incorporation of atmospheric oxygen. An unstable metabolite, 11-hydroxylinoleic acid, could also be isolated as well as (13R,13S)-hydroxy-(9E,9Z), (11E)-octadecadienoic acids and (9R,9S)-hydroxy-(10E), (12E,12Z)-octadecadienoic acids. In summary, G. graminis contains a prominent linoleic acid (8R)-dioxygenase, which differs from the lipoxygenase family of dioxygenases by catalyzing the formation of a hydroperoxide without affecting the double bonds of the substrate.