Biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid: identification of an allene oxide cyclase

Incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn (Zea mays L.) hydroperoxide dehydrase led to the formation of an unstable allene oxide derivative, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid. Further conversion of the allene oxide yielded two major products, i.e. alpha-ketol 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid, and 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA). 12-Oxo-PDA was formed from allene oxide by two different pathways, i.e. spontaneous chemical cyclization, leading to racemic 12-oxo-PDA, and enzyme-catalyzed cyclization, leading to optically pure 12-oxo-PDA. The allene oxide cyclase, a novel enzyme in the metabolism of oxygenated fatty acids, was partially characterized and found to be a soluble protein with an apparent molecular weight of about 45,000 that specifically catalyzed conversion of allene oxide into 9(S),13(S)-12-oxo-PDA.     

Pathways of Fatty Acid hydroperoxide metabolism in spinach leaf chloroplasts

The metabolism of 13-hydroperoxylinolenic acid was examined in protoplasts and homogenates prepared from mature leaves of spinach (Spinacia oleracea L.). Chloroplast membranes were the principal site for metabolism of the compound by at least two highly hydrophobic enzyme systems, hydroperoxide lyase and hydroperoxide dehydrase, the new name for an enzyme system formerly known as hydroperoxide isomerase and hydroperoxide cyclase. Hydroperoxide lyase was most active above pH 7 and could be separated from hydroperoxide dehydrase by anion exchange chromatography. Hydroperoxide dehydrase, measured by the formation of both α-ketol product and 12-oxo-phytodienoic acid, had its optimum activity in the range of pH 5 to 7. Lyase was more active than dehydrase activity when the enzymes were extracted by homogenization. The reverse was true when the enzyme activities were measured in protoplasts, which are isolated by gentle extraction methods. The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response. In the absence of injury, the normal pathway of fatty acid hydroperoxide metabolism is probably by hydroperoxide dehydrase activity. The molecular weights of both the lyase and dehydrase were approximately 220,000, as estimated by gel filtration.     

Fatty acid allene oxides. II. Formation of two macrolactones from 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

An unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid, was recently identified as the product formed from 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid in the presence of corn (Zea mays L.) hydroperoxide dehydrase (M. Hamberg (1987) Biochim. Biophys. Acta 920, 76-84). The present paper is concerned with the spontaneous decomposition of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in acetonitrile solution. Two major products were isolated and characterized, i.e. macrolactones 12-keto-9(Z)-octadecen-11-olide and 12-keto-9(Z)-octadecen-13-olide.     

Allene oxide cyclase: a new enzyme in plant lipid metabolism

The mechanism of the biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA) from 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid in preparations of corn (Zea mays L.) was studied. In the initial reaction the hydroperoxide was converted into an unstable allene oxide, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, by action of a particle-bound hydroperoxide dehydrase. A new enzyme, allene oxide cyclase, catalyzed subsequent cyclization of allene oxide into 9(S),13(S)-12-oxo-PDA. In addition, because of its chemical instability, the allene oxide underwent competing nonenzymatic reactions such as hydrolysis into alpha- and gamma-ketol derivatives as well as spontaneous cyclization into racemic 12-oxo-PDA. (+/-)-cis-12,13-Epoxy-9(Z)-octadecenoic acid and (+/-)-cis-12,13-epoxy-9(Z),15(Z)-octadecadienoic acid, in which the epoxy group was located in the same position as in the allene oxide substrate, served as potent inhibitors of corn allene oxide cyclase. On the other hand, the isomeric (+/-)-cis-9,10-epoxy-12(Z)-octadecenoic acid had little inhibitory effect. Allene oxide cyclase was present in the soluble fraction of corn homogenate and had a molecular weight of about 45,000 as judged by gel filtration. The enzyme activity was detected in several plant tissues, the highest levels being observed in potato tubers and in leaves of spinach and white cabbage.     

Purification of a lipoxygenase from ungerminated barley. Characterization and product formation

Lipoxygenase was purified from ungerminated barley (variety 'Triumph'), yielding an active enzyme with a pI of 5.2 and a molecular mass of approximately 90 kDa. In addition to the 90 kDa band SDS-PAGE showed the presence of two further proteins of 63 kDa. Western blot analysis showed cross-reactivity of each of these proteins with polyclonal antisera against lipoxygenases from pea as well as from soybean, suggesting a close immunological relationship. The 63 kDa proteins appear to be inactive degradation products of the active 90-kDa enzyme. This barley lipoxygenase converts linoleic acid mainly into (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid, and arachidonic acid into (5S)-(6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eic osatetraenoic acid.     

Hydroperoxide-dependent epoxidation of unsaturated fatty acids in the broad bean (Vicia faba L.)

Incubation of linoleic acid with the 105,000g particle fraction of the homogenate of the broad bean (Vicia faba L.) led to the formation of the following products: 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid, 9,10-epoxy-12(Z)-octadecenoic acid (9(R),10(S)/9(S)/10(R), 80/20), 12,13-epoxy-9(Z)-octadecenoic acid (12(S),13(R)/12(R)/13(S), 64/36), and 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid (9(S),10(R)/9(R),10(S), 91/9). Oleic acid incubated with the enzyme preparation in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid or cumene hydroperoxide was converted into 9,10-epoxyoctadecanoic acid (9(R),10(S)/9(S),10(R), 79/21). Two enzyme activities were involved in the formation of the products, an omega 6-lipoxygenase and a hydroperoxide-dependent epoxygenase. The lipoxygenase, but not the epoxygenase, was inhibited by low concentrations of 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid. In contrast, the epoxygenase, but not the lipoxygenase, was readily inactivated in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid. Studies with 18O2-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the epoxide oxygens of 9,10-epoxyoctadecanoic acid and of 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid were derived from hydroperoxide and not from molecular oxygen.     

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.     

Formation of prostaglandin A analogues via an allene oxide

One potential biosynthetic route to the prostaglandins involves the participation of lipoxygenase and allene oxide synthase enzymes, giving a hydroxylated allene oxide, which then might cyclize to form prostaglandin A or a close analogue. We have tested a model of this type of transformation using 8-hydroxy-15S-hydroperoxy eicosanoids as substrates for the dehydrase (allene oxide synthase) in flaxseed. Four of these substrates, each with a 9E,11Z,13E-conjugated triene, gave an observable rate of reaction. The two derived from eicosapentaenoic acid reacted more rapidly than the corresponding arachidonic acid analogues. Also, the 8S-hydroxy-15S-hydroperoxy diastereomers reacted more rapidly than their 8R-hydroxy analogues. Products were characterized by high pressure liquid chromatography, UV, gas chromatography-mass specrometry, 1H NMR, and CD. Reaction of the (8S)-hydroxy-(15S)-hydroperoxy-eicosapentaenoic acid gave two alpha-ketols [8S),15-dihydroxy-14-oxoeicosa-5Z,9E,11Z,17Z+ ++-tetraenoic acid and the corresponding 11E isomer in a 2:1 ratio), together with four prostaglandin A3 analogues which differed in the configurations of the side chains. Oxygen 18 labeling fully supported the intermediacy of an allene oxide in the biosynthesis. The corresponding "8R" substrate was converted to the enantiomers of these products plus three 13-hydroxy-14,15-epoxy derivatives. The arachidonate analogues formed the epoxy-hydroxy derivatives, the alpha-ketols, and two prostaglandin A2 analogues with trans configuration of the side chains. These results demonstrate (i) a feasible route of metabolism of lipoxygenase products to hydroxy allene oxide, (ii) the potential for the resulting allene oxide to cyclize to a prostaglandin A analogue, and (iii) the marked influence of the hydroxyl configuration of the rate of reaction and resulting profile of products. Some of these reactions may occur in a natural pathway of prostanoid biosynthesis in corals and other organisms.     

Evidence for participation of iron in lipoxygenase reaction from optical and electron spin resonance studies.

Optical and EPR studies indicate that the iron present in lipoxygenase participates in catalysis. Addition of linoleic acid hydroperoxide to lipoxygenase 1 causes an increase in abosrbance over the range of 350 to 650 nm which is reversed when linoleic acid hydroperoxide is destroyed upon the addition of linoleic acid under anaerobic conditions. Lipoxygenase 1 alone exhibits no EPR signal but upon addition of linoleic acid hydroperoxide or linoleic acid several signals appear. Addition of linoleic acid hydroperoxide results in an EPR signal at g approximately equal to 6 accompanied by a small but relatively sharp signal at g approximately equal to 2. Under anaerobic conditions the latter is replaced by a broad anisotropic signal around g approximately equal to 2. The appearance of the EPR signal at g approximately equal to 6 coincides with the change in the optical spectrum of the enzyme. When linoleic acid is added under anaerobic conditions a broad anisotropic EPR signal around g approximately equal to 2 is observed. Thus it appears that lipoxygenase can exist in two forms: (a) a resting form with a very weak absorbance in the visible range of the light spectrum and no EPR signal and (b) an active form (after addition of linoleic acid hydroperoxide) with an increased optical absorbance and EPR signal at g approximately equal to 6. This observation may be related to the earlier discovery that the lipoxygenase reaction occurs with a lag which can be overcome by addition of product hydroperoxide. The EPR experiments indicate that lipoxygenase in the active form contains high spin ferric ion. Although EPR signals in the g approximately equal to 6 region are frequently observed with heme proteins, the only nonheme protein, other than lipoxygenase, reported to show an EPR signal in this region is the phenolytic dioxygenase, protocatechuate 3,4-dioxygenase (Peisach, J., Fujisawa, H., Blumberg, W. E., and Hayaishi, O. (1972) Fed. Proc. 31, 448).     

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.     

Allene oxide and aldehyde biosynthesis in starfish oocytes.

Allene oxides are a very unusual type of epoxide that, in biological systems, are formed by the enzymic dehydration of fatty acid hydroperoxides (lipoxygenase products). This reaction occurs widely in plants, in which allene oxide synthesis is a key step in the conversion of linolenic acid to jasmonic acid, the plant growth regulator. We report biosynthesis of the allene oxide (8R)-8,9-epoxyeicosa-(5Z,9,11Z,14Z)-tetraenoic acid via the (8R)-lipoxygenase metabolism of arachidonic acid in starfish oocytes. Formation of the allene oxide was deduced from high pressure liquid chromatography, UV, gas chromatography-mass spectrometry and 1H-NMR analyses of the precise structure and mechanism of biosynthesis of its major hydrolysis product, the alpha-ketol 8-hydroxy-9-ketoeicosa-(5Z,11Z,14Z)-trienoic acid. A second enzymic activity detected in the oocytes (hydroperoxide lyase) cleaves specifically the (8R)-hydroperoxy substrate into C7 and C13 fragments, identified as the hydroxyacid, (5Z)-7-hydroxyheptenoic acid, and two aldehydes, (2E,4Z,7Z)-tridecenal and its 4E isomer. Discovery of the allene oxide synthase and hydroperoxide lyase marks the first definitive localization of these enzymic activities to an animal cell. It was established previously that the (8R)-lipoxygenase metabolite (8R)-HETE will activate the maturation (re-initiation of meiosis) of starfish oocytes. The individual 8-lipoxygenase products may be involved at distinct stages of cell development.     

Role of lipoxygenase in the O2-dependent activation of soluble guanylate cyclase from rat lung.

Guanylate cyclase activity in rat lung supernatant fractions is stimulated 3-4 fold by aerobic incubation at 30 degrees C for approx. 30 min ('O2-dependent activation'). This stimulation was blocked by 20 microM-eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of lipoxygenase and cyclo-oxygenase, but not by aspirin or indomethacin, which are cyclo-oxygenase inhibitors. The enzyme activator(s) is presumed to be the fatty acid hydroperoxide(s) formed by lipoxygenase. Removal of lipoxygenase from the supernatant fraction by chromatography on Amberlite XAD-4 also prevented activation, which was restored by the addition of soya-bean lipoxygenase. Bovine serum albumin prevented O2-dependent activation or activation by soya-bean lipoxygenase, through its ability to bind the unsaturated fatty acid substrate of lipoxygenase. The lipoxygenase in the supernatant fraction is inhibited by endogenous glutathione peroxidase plus reduced glutathione (GSH); removal of GSH de-inhibits lipoxygenase and activates guanylate cyclase. This was effected by autoxidation, by cumene hydroperoxide (with GSH peroxidase) and by titration with N-ethylmaleimide (NEM). Activation by NEM was inhibited by serum albumin or ETYA, as was activation by low concentrations (less than 50 microM) of cumene hydroperoxide. Activation by higher concentrations was not so inhibited; therefore, cumene hydroperoxide can also activate by a direct effect on guanylate cyclase. A hypothesis for physiological activation is proposed.     

Biosynthesis of C9-aldehydes in the moss Physcomitrella patens

After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.     

Formation of a new class of oxylipins from N-acyl(ethanol)amines by the lipoxygenase pathway.

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both alpha-N-linolenoylethanolamine and gamma-N-linolenoylethanolamine (18:3), as well as alpha-N-linolenoylamine and gamma-N-linolenoylamine were converted into their (13S)-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of alpha-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z)-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)-hydroperoxy-alpha-N-linolenoylethanolamine and the (13S)-hydroperoxide of linoleic acid and its ethanolamine derivative. alpha-Ketols and gamma-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S)-hydroperoxy-alpha-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.     

Influence of (9Z)-12-hydroxy-9-dodecenoic acid and methyl jasmonate on plant protein phosphorylation

The products of the lipoxygenase pathway, methyl jasmonic acid (MeJA) and (9Z)-12-hydroxy-9-dodecenoic acid (HDA), hardly changed the relative level of phosphorylated polypeptides (RLPPs) during 2 h of incubation: 15 and 17 kDa RLPPs were enhanced by HDA, but decreased by MeJA. RLPPs of 73 and 82 kDa were increased by both compounds. MeJA and HDA treatment induced specific and unspecific effects in some RLPPs. It was shown that HDA and MeJA increased protein kinase activity in the presence of 1 microM cAMP.     

Characterization of 9-fatty acid hydroperoxide lyase-like activity in germinating barley seeds that transforms 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid into 2(E)-nonenal

Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r=0.53, P<0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.     

Factors affecting the initial rate of lipoxygenase catalysis

The rate of peroxidation of linoleic acid by soybean type-1 lipoxygenase was studied under conditions which assured that the substrate was present as a monomolecular solution and that the first 5% of the reaction was observed. In order to achieve this, the kinetics were carried out at pH 10.0 in borate buffer using linoleic acid and enzyme concentrations of less than 75 μM and 0.2 nM respectively. The initial rate was increased by the presence of added product (13-hydroperoxy-9(Z),11(E)-octadecadienoic acid) in the substrate solutions in a concentration dependent and saturatable fashion. Product analogues lacking the hydroperoxide group (13-hydroxy-9(Z),11(E)-octadecadienoic acid and 13-methoxy-9(Z),11(E)-octadecadienoic acid) did not evoke this rate enhancing effect. These compounds reduced the initial rate when preincubated with enzyme prior to mixing with substrate. The results indicated that the chemical reactivity of the product was a necessary requirement for its activating effect on the enzyme.     

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.     

Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase

Manganese lipoxygenase (Mn-LOX) catalyzes the rearrangement of bis-allylic S-hydroperoxides to allylic R-hydroperoxides, but little is known about the reaction mechanism. 1-Linoleoyl-lysoglycerophosphatidylcholine was oxidized in analogy with 18:2n-6 at the bis-allylic carbon with rearrangement to C-13 at the end of lipoxygenation, suggesting a "tail-first" model. The rearrangement of bis-allylic hydroperoxides was influenced by double bond configuration and the chain length of fatty acids. The Gly316Ala mutant changed the position of lipoxygenation toward the carboxyl group of 20:2n-6 and 20:3n-3 and prevented the bis-allylic hydroperoxide of 20:3n-3 but not 20:2n-6 to interact with the catalytic metal. The oxidized form, Mn(III)-LOX, likely accepts an electron from the bis-allylic hydroperoxide anion with the formation of the peroxyl radical, but rearrangement of 11-hydroperoxyoctadecatrienoic acid by Mn-LOX was not reduced in D(2)O (pD 7.5), and aqueous Fe(3+) did not transfer 11S-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid to allylic hydroperoxides. Mutants in the vicinity of the catalytic metal, Asn466Leu and Ser469Ala, had little influence on bis-allylic hydroperoxide rearrangement. In conclusion, Mn-LOX transforms bis-allylic hydroperoxides to allylic by a reaction likely based on the positioning of the hydroperoxide close to Mn(3+) and electron transfer to the metal, with the formation of a bis-allylic peroxyl radical, beta-fragmentation, and oxygenation under steric control by the protein.