Peroxygenase-Catalyzed Fatty Acid Epoxidation in Cereal Seeds (Sequential Oxidation of Linoleic Acid into 9(S),12(S),13(S)-Trihydroxy-10(E)-Octadecenoic Acid)

Peroxygenase-catalyzed epoxidation of oleic acid in preparations of cereal seeds was investigated. The 105,000g particle fraction of oat (Avena sativa) seed homogenate showed high peroxygenase activity, i.e. 3034 [plus or minus] 288 and 2441 [plus or minus] 168 nmol (10 min)-1 mg-1 protein in two cultivars, whereas the corresponding fraction obtained from barley (Hordeum vulgare and Hordeum distichum), rye (Secale cereale), and wheat (Triticum aestivum) showed only weak activity, i.e. 13 to 138 nmol (10 min)-1 mg-1 protein. In subcellular fractions of oat seed homogenate, peroxygenase specific activity was highest in the 105,000g particle fraction, whereas lipoxygenase activity was more evenly distributed and highest in the 105,000g supernatant fraction. Incubation of [1-14C]linoleic acid with the 105,000g supernatant of oat seed homogenate led to the formation of several metabolites, i.e. in order of decreasing abundance, 9(S)-hydroxy-10(E),12(Z)-octadecadienoic acid, 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, cis-9,10-epoxy-12(Z)-octadecenoic acid [mainly the 9(R),10(S) enantiomer], cis-12,13-epoxy-9(Z)-octadecenoic acid [mainly the 12(R),13(S) enantiomer], threo-12,13-dihydroxy-9(Z)-octadecenoic acid, and 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. Incubation of linoleic acid with the 105,000g particle fraction gave a similar, but not identical, pattern of metabolites. Conversion of linoleic acid into 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, a naturally occurring oxylipin with antifungal properties, took place by a pathway involving sequential catalysis by lipoxygenase, peroxygenase, and epoxide hydrolase.     

Epoxidation of fatty acids,fatty methyl esters,and alkenes by immobilized oat seed peroxygenase

Fatty epoxides are used as plasticizers and plastic stabilizers and are intermediates for the production of other chemical substances. The currently used industrial procedure for fatty epoxide synthesis requires a strong acid catalyst which can cause oxirane ring opening and side product formation. To find a replacement for the acid catalyst, we have been conducting research on a peroxygenase enzyme from oat (Avena sativa) seeds and have devised a method for immobilization of this enzyme using a hydrophobic membrane support. In this study, fatty acids and fatty methyl esters commonly encountered in commercial vegetable oils were tested as substrates for immobilized peroxygenase, and the epoxide products were characterized. The epoxidation time course of linoleic acid showed two distinct phases with nearly complete conversion to monoepoxide before diepoxide was produced. The diepoxide formed from linolenic acid was found to be 9,10-15,16-diepoxy-12-octadecenoic acid, and only a trace of triepoxide was obtained. Additionally it was discovered that acyclic alkenes with internal double bonds, a cyclic alkene, and an alkene with an aromatic substituent were substrates of peroxygenase. However, alkenes with terminal unsaturation were unreactive. With every substrate examined, oat seed peroxygenase exhibited specificity for epoxidation, producing no other products, and oxirane ring opening did not occur.     

Purification and Characterization of Allene Oxide Cyclase from Dry Corn Seeds

Allene oxide cyclase (AOC; EC 5.3.99.6) catalyzes the cyclization of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid to 12-oxo- 10,15(Z)-phytodienoic acid, the precursor of jasmonic acid (JA). This soluble enzyme was purified 2000-fold from dry corn (Zea mays L.) kernels to apparent homogeneity. The dimeric protein has a molecular mass of 47 kD. Allene oxide cyclase activity was not affected by divalent ions and was not feedback-regulated by its product, 12-oxo-l0,15(Z)-phytodienoic acid, or by JA. ([plus or minus])-cis- 12,13-Epoxy-9(Z)-octadecenoic acid, a substrate analog, strongly inhibited the enzyme, with 50% inhibition at 20 [mu]M. Modification of the inhibitor, such as methylation of the carboxyl group or a shift in the position of the epoxy group, abolished the inhibitory effect, indicating that both structural elements and their position are essential for binding to AOC. Nonsteroidal anti-inflammatory drugs, which are often used to interfere with JA biosynthesis, did not influence AOC activity. The purified enzyme catalyzed the cyclization of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid derived from linolenic acid, but not that of 12,13(S)-epoxy-9(Z),11- octadecadienoic acid derived from linoleic acid.     

Conversion of linoleic acid hydroperoxide to hydroxy, keto, epoxyhydroxy, and trihydroxy fatty acids by hematin

We have carried out a study of the reaction of 13-hydroperoxy-9-cis,11-trans-octadecadienoic acid (linoleic acid hydroperoxide) with hematin. The major products are erythro-11-hydroxy-12,13-epoxy-9-octadecenoic acid, threo-11-hydroxy-12,13-epoxy-9-octadecenoic acid, 9,12,13-trihydroxy-10-octadecenoic acid, 13-keto-9,11-octadecadienoic acid, and 13-hydroxy-9,11-octadecadienoic acid. Several minor products have also been identified, including 9-hydroxy-12,13-epoxyoctadecenoic acid, 11-hydroxy-9,10-epoxy-12-octadecenoic acid, 9-hydroxy-10,12-octadecadienoic acid, and 9-keto-10,12-octadecadienoic acid. Oxygen labeling studies indicate that the observed products arise by at least two pathways. In the major pathway, hematin reduces 13-hydroperoxy-9,11-octadecadienoic acid by one electron to an alkoxyl radical that cyclizes to an adjacent double bond to form an epoxy allylic radical. The allylic radical either couples to the hydroxyl radical coordinated to hematin or diffuses from the solvent cage and couples to O2, forming a peroxyl radical. In the minor pathway, the hydroperoxide is oxidized by one electron to a 13-peroxyl radical that undergoes beta-scission to a pentadienyl radical and O2. Exchange of hydroperoxide-derived O2 for dissolved O2 occurs at this stage followed by coupling of O2 to either terminus of the pentadienyl radical. Both pathways of hydroperoxide metabolism generate significant quantities of peroxyl radicals that epoxidize the isolated double bonds of dihydroaromatic molecules. The products of hydroperoxide reaction with hematin and the oxygen labeling patterns are very similar to the products of unsaturated fatty acid hydroperoxide metabolism by platelets, aorta, and lung. Our results not only provide a mechanism for the formation of a series of mammalian metabolites of linoleic and arachidonic acids but also offer an estimate of the yield of peroxyl radicals generated during the process.     

Stereochemistry of the epoxidation of fatty acids catalyzed by soybean peroxygenase

The stereochemistry of C18 unsaturated fatty acids epoxidation catalyzed by detergent-solubilized and partially purified soybean peroxygenase was determined by chiral phase HPLC. Linoleic acid was oxidized into 9, 10- and 12,13-cis-epoxyoctadecenoic acids with a high enantiofacial selectivity. A 5.2:1 and 2.3:1 ratio respectively in favor of the 9(R), 10(S)- and 12(R), 13(S)-epoxy enantiomers was observed. These epoxy-derivatives of linoleic acid have the chirality of metabolites known to be involved in plant defense against fungi. This finding is of importance in establishing a physiological role for the peroxygenase.     

Efficient epoxidation of unsaturated fatty acids by a hydroperoxide-dependent oxygenase

Detergent-solubilized and partially purified soybean peroxygenase was shown to actively catalyze, in the presence of alkylhydroperoxides as co-substrates, the epoxidation of mono- and polyunsaturated fatty acids such as oleic and linoleic acids. Octadecenoic acids were found to be better substrates than shorter mono-unsaturated fatty acids (C16:1 or C14:1), but the position of their double bond (at positions 6, 9, or 11) had little effect on the rates of epoxidation. The peroxygenase exhibits a strong stereospecificity since octadecenoic acids with double bonds in trans-configuration were not epoxidized at detectable rates. Oxidation of linoleic acid yielded the two positional monoepoxide isomers and, as the minor product, the diepoxide. An important regioselectivity was, however, observed in this case; i.e. the unsaturation at position 9,10 was epoxidized preferentially to that at 12, 13. Oxidation of oleic acid in the presence of 18O-labeled hydroperoxy-linoleic acid revealed an incorporation of about 80% of the label into the epoxide ring. Products similar to those formed by the peroxygenase by epoxidation of unsaturated free fatty acids such as linoleic acid have been described as important metabolites (leukotoxins) in the defense of plants, e.g. in fungal agressions. This aspect underlines the physiological relevance of this new and potent catalytic activity of the peroxygenase.     

Oxygenation of trans polyunsaturated fatty acids by lipoxygenase reveals steric features of the catalytic mechanism

Lipoxygenase, a nonheme iron dioxygenase, catalyzes the oxygenation of 1,4-diene units in polyunsaturated fatty acids, forming conjugated diene hydroperoxides as the primary products. The naturally occurring all-Z geometry for the olefins in the polyunsaturated fatty acid has long been thought to be a substrate requirement for the enzyme. A rigorous test of this hypothesis using the two isomeric (9E,12Z)- and (9Z,12E)-9,12-octadecadienoic acids was carried out. Both isomeric substrates were found to be catalytically oxygenated by soybean lipoxygenase 1 at a significant fraction of the rate of the reaction of the natural substrate, linoleic acid. Product determinations revealed that a thermodynamically unfavorable E to Z isomerization at the 9,10-position occurred when (9E,12Z)-9,12-octadecadienoic acid was converted into the 13-hydroperoxide by lipoxygenase 1. Determination of the stereochemistry at the oxygenated position in the products indicated that a comparable isomerization at the 12,13-position did not occur when the 9Z,12E isomer was employed. The distribution of products obtained from oxygenation at the 9-position supported the hypothesis that the enzyme catalyzes the reaction in one of two substrate orientations, conventional and head to tail reversed. The observations can be understood on the basis of the steric demands on intermediates in the proposed mechanism of action as well as by catalysis by the active-site iron atom.     

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.     

On the Specificity of a Fatty Acid Epoxygenase in Broad Bean (Vicia faba L.)

Seeds of broad bean (Vicia faba L.) contain a hydroperoxide-dependent fatty acid epoxygenase. Hydrogen peroxide served as an effective oxygen donor in the epoxygenase reaction. Fifteen unsaturated fatty acids were incubated with V. faba epoxygenase in the presence of hydrogen peroxide and the epoxy fatty acids produced were identified. Examination of the substrate specificity of the epoxygenase using a series of monounsaturated fatty acids demonstrated that (Z)-fatty acids were rapidly epoxidized into the corresponding cis-epoxy acids, whereas (E)-fatty acids were converted into their trans-epoxides at a very slow rate. In the series of (Z)-monoenoic acids, the double bond position as well as the chain length influenced the rate of epoxidation. The best substrates were found to be palmitoleic, oleic, and myristoleic acids. Steric analysis showed that most of the epoxy acids produced from monounsaturated fatty acids as well as from linoleic and α-linolenic acids had mainly the (R),(S) configuration. Exceptions were C₁₈ acids having the epoxide group located at C-12/13, in which cases the (S),(R) enantiomers dominated. 13(S)-Hydroxy-9(Z),11(E)-octadecadienoic acid incubated with epoxygenase afforded the epoxy alcohol 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as the major product. Smaller amounts of the diastereomeric epoxy alcohol 9(R),10(S)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid as well as the α,β-epoxy alcohol 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid were also obtained. The soluble fraction of homogenate of V. faba seeds contained an epoxide hydrolase activity that catalyzed the conversion of cis-9,10-epoxyoctadecanoic acid into threo-9,10-dihydroxyoctadecanoic acid.     

Oxygenation of linolenic and linoleic acid to novel vicinal dihydroxy acids by hepatic microsomes of the rabbit

[14C] Linolenic acid (18: omega 3) and [14C] linoleic acid (18:2 omega 6) were incubated with hepatic microsomes of the rabbit in the presence of NADPH (1 Mm) for 15 min at 37 degrees C. The products were extracted and purified by high performance liquid chromatography. The major metabolites of linolenic and linoleic acid were identified by capillary gas chromatography mass spectrometry as 15,16-dihydroxy-9,12-octadecadienoic acid, 12,13-dihydroxy-9,15-octadecadienoic acid and 9,10-dihydroxy-12,15-octadecadienoic acid and as 12,13-dihydroxy-9-octadecaenoic acid and 9,10-dihydroxy-12-octadecaenoic acid, respectively. The results were confirmed by comparison with mass spectra of the authentic compounds. These metabolites are presumably formed by cytochrome P-450 catalyzed epoxidation of the omega 3, omega 6 and omega 9 double bonds, followed by enzymatic hydrolysis to 1,2-diols. The ratio of omega 3, omega 6 and omega 9 oxygenated metabolites of linolenic acid was approximately 2:1:1 and the ratio of the omega 6 and omega 9 metabolites of linoleic acid was 2:1, indicating that the double bond closest the omega end is most easily oxygenated.     

Biosynthesis of cutin monomers: involvement of a lipoxygenase/peroxygenase pathway

Cutin is synthesized from oxygenated fatty acids derived preponderantly from oleic acid. The enzymatic pathways involved in the biosynthesis of such cutin monomers have been studied, i.e. 18-hydroxyoleic acid, 9,10-epoxy-18-hydroxystearic acid (the major constituent) and 9,10,18-trihydroxystearic acid. This was approached by studying (i) the substrate specificity and stereoselectivity of purified peroxygenase, which epoxidizes unsaturated fatty acids, and fatty acid epoxide hydrolase, i.e. two enzyme activities that have been found recently in higher plants, and (ii) the transformation of oleic acid into cutin monomers by a cell free system, i.e. soybean microsomes. These two enzymes, along with a ω-hydroxylating activity, can account for the biosynthesis of the oleic acid-derived cutin monomers and their precursors. A new biosynthetic scheme is proposed, whose pathways take into account the dynamic aspects of the expression of the different enzyme activities involved. Importantly, since peroxygenase, for its activity, is strictly dependent on fatty acid hydroperoxides, which act as co-substrates, the biosynthesis of cutin monomers is also dependent on the activity of lipoxygenases.     

Gas--liquid chromatography--mass spectrophotometry of hydroxylated octadecanols derived from hydroxylated stearic acids

A gas-liquid chromatographic-mass spectrometric method of determining the position of oxygen atoms on polyfunctional fatty acids has been explored. The method consists of reduction of keto, hydroperoxy, epoxy, and carboxylic acid groups with LiAlH(4) to the corresponding alcohols; trimethylsilylation with bis(trimethylsilyl)acetamide; and analysis by means of the combined gas-liquid chromatograph-mass spectrometer. The following compounds were analyzed: 9-mono-, 9,10-di-, 9,10,12-tri-, and 9,10,12,13-tetrahydroxystearic acids and the corresponding derivatives of octadecan-1-ol. The reduction products of 9,10-epoxystearic acid and a mixture of linoleic acid 9- and 13-hydroperoxides were also analyzed. The position of the oxygen function in the original molecule can be deduced rapidly and accurately.     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

Regio- and enantioselectivity of soybean fatty acid epoxide hydrolase.

Soluble epoxide hydrolase purified from soybean catalyzes trans-addition of water across the oxirane ring of cis-9,10-epoxystearic acid with inversion of configuration at the attacked carbon, yielding threo-9,10-dihydroxystearic acid. Kinetic analyses of the progress curves, obtained at low substrate concentrations (i.e. [S] much less than Km), and determination of the enantiomeric excess of the residual substrate by chiral-phase high-performance liquid chromatography at different reaction times, indicate that the epoxide hydrolase hydrates preferentially cis-9R, 10S-epoxystearic acid (V/Km ratio, approximately 20). Interestingly, this enantiomer is obtained by epoxidation of oleic acid catalyzed by peroxygenase, a hydroperoxide-dependent oxidase, we have previously described in soybean (Blée, E., and Schuber, F. (1990) J.Biol. Chem. 265, 12887-12894). For the epoxide hydrolase to show high enantioselectivity there must be a free carboxylic acid functionality on the substrate which probably influences its positioning within the active site. This selectivity, which in principle can be used for kinetic resolution of the cis-9,10-epoxystearic acid enantiomers, is much reduced with methyl cis-9,10-epoxystearate. 18O-Labeling experiments indicate that water attacks both cis-9,10-epoxystearic acid enantiomers on the oxirane carbon which has the S-chirality. Results show that soybean epoxide hydrolase produces exclusively threo-9R,10R-dihydroxystearic acid, i.e. a naturally occurring metabolite in higher plants. cis-9,10-Epoxy-18-hydroxystearic acid, a cutin monomer, was a poorer substrate of the epoxide hydrolase than 9,10-epoxystearic acid (V/Km ratio for the preferred enantiomers, approximately 19). From a physiological point of view, peroxygenase and this newly described epoxide hydrolase could be responsible, in vivo, for the biosynthesis of a class of oxygenated fatty acid compounds known to be involved in cutin monomers production and in plant defense mechanisms.     

Syntheses and reactions of methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate

The syntheses and reactions of two epoxyketoacids (methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (IV) and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (V)) are described. The synthetic method is based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide. Reduction of the hydroperoxide with sodium borohydride followed by oxidation, esterification and epoxidation yielded the compounds IV and V with a global yield of 14% and 3%, respectively, referred to the diasteromerically pure isolated compounds. Confirmation of the structures was carried out by reduction of the ketone group with sodium borohydride and by the opening of the oxirane ring with methanolic boron trifluoride. The reduction of compounds IV and V with hydrogen mainly yielded the tetrahydrofuranoid fatty acid, methyl 10,13-epoxyoctadecanoate. This reaction may be considered a new procedure to obtain tetrahydrofuranoid fatty acids.     

Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 2: mass spectral characterization

Methyl esters of C18 polyunsaturated fatty acids, including gamma-linolenic acid, alpha-linolenic acid and stearidonic acid, were epoxidised using m-chloroperbenzoic acid. Nine monoepoxides were obtained by normal-phase HPLC, identified using LC-MS and NMR, and characterized by NMR spectroscopy and mass spectrometry. This study is focused on structural characterization using LC-MS and LC/APCI/MS/MS. The elution profiles of these monoepoxides in RP-HPLC are determined as 12,13->9,10->6,7-epoxy, 9,10->15,16->12,13-epoxy and 15,16->12,13->9,10-epoxy derivatives of gamma-linolenic, alpha-linolenic and stearidonic acid methyl esters, respectively. The major diagnostic fragmentations in MS/MS identified are postulated to be induced by cleavages of the epoxide ring and alpha-bond cleavage to the epoxy group from [M+H]+ and/or [M+H-MeOH]+.     

The effect of methyl jasmonate on free fatty acids content in ripening tomato fruits

The effect of methyl jasmonate (JA-Me), applied to mature green tomato fruits cv. Modena, on the content of some fatty acids in ripe fruits was studied. Methyl jasmonate greatly increased content of linolenic acid and in the lesser degree decreased the amount of linoleic acid. The ratio of linolenic acid to linoleic acid content increased 4.5–7.7 times in methyl jasmonate treated samples in comparison to untreated-controls. JA-Me did not affect the contents of lauric, myristic, palmitic, stearic, palmitoleic and oleic acids.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

Oxygenation of unsaturated fatty acids in seeds during storage

Seeds of Cichorium intybus L., Crepis thomsonii Babc, and Crepis vesicaria L, were stored from 4 to 8 years at 5°C and then for 18 months under a variety of conditions. Oxygenated acids in Cichorium intybus oil increased from approximately 1% initially to 3% in the first storage period and to 17% while stored at room temperature during the second period. The corresponding levels at these three stages for Crepis thomsonii were 2, 6 and 18%. By gas chromatography (GC) and GC-mass spectrometry, the major oxygenated acids formed during storage were identified as hydroxy acids with conjugated unsaturation and 9,10-epoxy acids. In Crepis vesicaria seed, oil of which contained 53% vernolic (12,13-epoxy-9-octadecenoic) acid originally, approximately 2% of 9,10-epoxides were formed during the storage at room temperature. Levels of hydroxy acids with conjugated unsaturation in this species were 0.3% initially, 2% after 5 years at 5°C, and 9% after 18 months at room temperature. Primary substrates from which oxygenated acids were formed in the three species were crepenynic and linoleic acids, and the almost exclusive formation of 9,10-epoxide from linoleic acid indicated enzymatic involvement.     

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.