Hydroperoxide Dehydrase Research Articles

Fatty acid hydroperoxides pathways in plants. A review.

The present paper focusses on the fatty acid hydroperoxides pathways, mainly hydroperoxide lyase and hydroperoxide dehydrase. For each enzyme, the definition, occurrence and subcellular localization is presented. Particular attention is given to reaction mecanisms and to substrate specificity. Physiological roles of reaction products are also discussed.

Formation of epoxyalcohols by a purified allene oxide synthase. Implications for the mechanism of allene oxide synthesis.

The allene oxide synthase (hydroperoxide dehydrase) of flaxseed is a cytochrome P450 that exhibits an exceptionally high catalytic turnover (> or = 1000/s) for hydroperoxy substrates. In a previous study, using a crude extract of flaxseed, we detected a secondary activity that could offer an insight into the mechanism of the enzymatic transformation of hydroperoxides. We observed that the substrate 8R-hydroxy-15S-hydroperoxyeicosa-5,9,11,13,17-pentaenoic acid is converted not only to allene oxide, but also to epoxyalcohol derivatives (Brash, A. R., Baertschi, S. W., and Harris, T. M. (1990) J. Biol. Chem. 265, 6705-6712). The transformation of hydroperoxides to epoxyalcohols has been investigated extensively in other systems, and heterolytic or homolytic cleavage of the hydroperoxide is associated with characteristic rearrangements and stereochemistry of the epoxyalcohol products. Using the purified enzyme, we established that the epoxyalcohols are products of the allene oxide synthase. Their structures were determined by UV, gas chromatography-mass spectrometry, and NMR. The major epoxyalcohol is 8R,13R-dihydroxy-14R,15S-epoxyeicosa-5Z,9E ,11Z,17Z-tetraenoic acid, a trans-epoxide with an alpha-hydroxyl in the relative threo configuration. Two minor products are the corresponding 11E isomer and a cis-epoxide identified as 8R,13-dihydroxy-14S,15S-epoxyeicosa-5Z,9E,11E,++ +17Z-tetraenoic acid. Gas chromatography-mass spectrometry analysis of a reaction with [18O2]hydroperoxide substrate indicated complete retention of the hydroperoxy oxygens in the epoxyalcohol products. Mechanistic precedents support a homolytic hydroperoxide cleavage as the initial step in the synthesis of these epoxyalcohols. We suggest that the same process initiates allene oxide synthesis, a conclusion that is also most compatible with the known chemistry of cytochromes P450.

Purification of lipoxygenase and hydroperoxide dehydrase in flaxseeds: Interaction between these enzymatic activities

We have purified two enzymic activities from flaxseed acetone powder : a lipoxygenase and a hydroperoxide dehydrase. The lipoxygenase activity belongs to an iron-containing protein having a molecular weight of 130 kDa which, upon incubation with α-linolenic acid, forms 13-hydroperoxy-9(Z), 11 (E), 15(Z)- octadecatrienoic acid. The hydroperoxide dehydrase (a 55 kDa protein) metabolizes this hydroperoxide to an allene oxide which in turn is spontaneously hydrolyzed to α-and γ-ketols. Relationships between these two enzymes were studied and results suggest an inhibition of the lipoxygenase by hydroperoxide dehydrase.

Formation of ketols from linolenic acid 13-hydroperoxide via allene oxide. Evidence for two distinct mechanisms of allene oxide hydrolysis

Incubations of [1-14C]13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) with hydroperoxide dehydrase preparations from flax seeds lead to the formation of a novel ketol 2 along with the previously known 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic (12,13-alpha-ketol) and 9-hydroxy-12-oxo-10(E),15(Z)-octadecadienoic (gamma-ketol) acids. Compound 2 was identified as 11-hydroxy-12-oxo-9(Z),15(Z)-octadecadienoic acid (11,12-alpha-ketol) in accordance with the data of ultraviolet, mass (chemical ionization and electron impact) and 1H-NMR spectra. During long-term (30 min) incubations the yields of gamma-ketol and 11,12-alpha-ketol increased markedly and the yield of 12,13-alpha-ketol decreased in response to the pH change from basic (pH 7.4) to acidic (pH 5.8) conditions. Short-term (15 s) incubations of 13-HPOT with hydroperoxide dehydrase, terminated by HCl fixation, led to the formation of gamma-ketol and ketol 2. A similar incubation, followed by NaOH fixation, afforded only 12,13-alpha-ketol. The trapping of allene oxide (a primary product of hydroperoxide dehydrase) with pure methanol gives only compound 4 (12,13-alpha-ketol methyl ether). Products 5 (gamma-ketol methyl ether) and 6 (11,12-alpha-ketol methyl ether) were formed along with 4 as a result of trapping with acidified methanol. The results obtained indicate that: (a) the formation of 12,13-alpha-ketol is base-dependent; (b) the formation of gamma-ketol and ketol 2 is acid-dependent. Two distinct mechanisms of allene oxide hydrolysis are proposed: (1) nucleophilic (SN2 or SN1, OH- is an attacking group) substitution, resulting in formation of 12,13-alpha-ketol; (2) electrophilic (SE-like) reaction initiated by protonation of oxirane, affording gamma-ketol and 11,12-alpha-ketol.

Hydroperoxides of α‐ketols

Metabolism of [1-14C]linolenic acid, 13-hydroperoxy-8(Z),11(E),15(Z)-[1-14C]octadecatrienoic acid (13-HPOT) and 9-hydroperoxy-10(E),12(Z),15(Z)-[1-14C]octadecatrienoic acid (9-HPOT) was studied by enzyme preparations from flax, wheat and corn seeds, containing two enzymes of fatty acid metabolism, namely, lipoxygenase and hydroperoxide dehydrase. Along with the previously known products of the hydroperoxide dehydrase pathway, the radiolabel was incorporated into some more polar metabolites. These polar products 1 and 4, formed from 13-HPOT and 9-HPOT, respectively, were purified by reversed-phase and normal-phase HPLC, and investigated by ultraviolet spectroscopy, chemical-ionization and electron-impact mass spectrometry, and 1H-NMR. The data obtained suggest that metabolites 1 and 4 are 9-hydroperoxy-12-oxo-13-hydroxy-10(E),15(Z)-octadecadienoic acid and 9-hydroxy-10-oxo-13-hydroperoxy-11(E),15(Z)-octadecadienoic acid, respectively. 12-oxo-13-hydroxy-9(Z),15(Z)-[1-14C]octadecadienoic acid (12,13-alpha-ketol) and 9-hydroxy-10-oxo-12(Z),15(Z)-[1-14C]octadecadienoic acid (9,10-alpha-keto) are the direct precursors of metabolites 1 and 4, respectively. Metabolites 1 and 4 are formed from the corresponding HPOT precursors in two stages; (a) conversion of hydroperoxide into the alpha-ketol by hydroperoxide dehydrase and (b) the lipoxygenase oxidation of the alpha-ketol. Different lipoxygenases were found to oxidize alpha-ketols. Oxidation of the 3(Z)-buten-1-onyl moiety of alpha-ketols presents an unusual and previously unknown type of lipoxygenase reaction.

Alteraciones bioquímicas de los lípidos en los alimentos vegetales. II. Metabolismo de los hidroperóxidos lipídicos

The enzymes of the lipoxygenase pathway in plant foods are reviewed. This part analyzes the enzymes related to the descomposition or transformation of the lipidic hydroperoxides. Lipoxygenase, hydroperoxide lyase, enal isomerase, alcohol dehydrogenase, aldehyde dehydrogenase, hydroperoxide dehydrase, alene oxide ciclase and 12-Oxo-phytodienoic acid reductase in plants are described. Posible control mechanisms of the lipoxygenase pathway are also considered.

A spectrophotometric assay for hydroperoxide lyase

AbstractSpectrophotometric assays for hydroperoxide lyase have traditionally measured the loss in absorption at 234 nm due to the disruption of conjugated diene in the fatty acid hydroperoxide. However, that assay does not distinguish between hydroperoxide lyase and hydroperoxide dehydrase activities, both of which cause a loss of conjugation in the substrate hydroperoxide. A new assay has been developed which is specific for hydroperoxide lyase. It is based on the ability of hydroperoxide lyase products, aldehydes and ω‐oxoacids, to serve as substrates for yeast alcohol dehydrogenase. Thus, the new hydroperoxide lyase assay is a coupled enzyme assay, which is conducted spectrophotometrically at 340 nm and measures the oxidation of nicotinamide adenenine dinucleotide, reduced form (NADH). In addition to its specificity for hydroperoxide lyase, the coupled assay allows higher concentrations of both fatty acid hydroperoxide substrate and crude enzyme extracts than the assay conducted at 234 nm.

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 particlebound 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 α- and γ-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.

Metabolism of Fatty Acid Hydroperoxides by Chlorella pyrenoidosa

The green alga Chlorella pyrenoidosa was examined for its ability to metabolize 13-hydroperoxylinoleic and 13-hydroperoxylinolenic acids. The study showed that Chlorella extracts possessed hydroperoxide dehydrase and other enzymes of the jasmonic acid pathway. However, under normal laboratory conditions for culture growth, neither jasmonic acid nor metabolites of the jasmonic acid pathway were present in Chlorella. In vitro enzyme studies also revealed the presence of hydroperoxide lyase activity that cleaved 13-hydroperoxylinoleic or 13-hydroperoxylinolenic acid into two products, 13-oxo-cis-9,trans-11-tridecadienoic acid and pentane (from linoleic acid) or pentene (from linolenic acid). The lyase was heat-labile, insensitive to 50 millimolar KCN, and had an approximate molecular weight of 48,000 as estimated by gel filtration. Two other products, 13-hydroxy-cis-9,trans-11,cis-15-octadecatrienoic acid and 12, 13-trans-epoxy-9-oxo-trans-10,cis-15-octadecadienoic acid, were also observed. Because these compounds are also products of nonenzymic, Fe(II)-catalyzed hydroperoxide decomposition reactions, their presence suggested that the observed lyase activity may occur via a homolytic decomposition mechanism.

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.

Absolute configuration of 12‐Oxo‐10,15(Z)‐phytodienoic acid

Abstract12‐Oxo‐10,15(Z)‐phytodienoic acid biosynthesized from 13(S)‐hydroperoxy‐9(Z),11(E),15(Z)‐octadecatrienoic acid using a preparation of corn (Zea mays L) hydroperoxide dehydrase recently was found to be a mixture of enantiomers in a ratio of 82∶18 (Hamberg, M., and Hughes, M.A. (1988)Lipids 23, 469–475). In this work, 12‐oxophytodienoic acid and (+)‐7‐iso‐jasmonic acid were converted into a common derivative, methyl 3‐hydroxy‐2‐pentyl‐cyclopentane‐1‐octanoate. From gas liquid chromatographic analysis of the (−)‐menthoxycarbonyl derivative of methyl 3‐hydroxy‐2‐pentyl‐cyclopentane‐1‐octanoates prepared from 12‐oxophytodienoic acid and (+)‐7‐iso‐jasmonic acid, it could be deduced that the major enantiomer of 12‐oxophytodienoic acid had the 9(S),13(S) configuration. Therefore, in the major enantiomer of 12‐oxophytodienoic acid, the configurations of the side chainbearing carbons are identical to the configurations of the corresponding carbons of (+)‐7‐iso‐jasmonic acid, thus giving support to previous studies indicating that 12‐oxophytodienoic acid serves as the precursor of (+)‐7‐iso‐jasmonic acid in plant tissue.

Fatty acid allene oxides. III. Albumin‐induced cyclization of 12,13(S)‐epoxy‐9(Z),11‐octadecadienoic acid

AbstractRecently, corn (Zea mays L.) hydroperoxide dehydrase was found to catalyze the conversion of 13(S)‐hydroperoxy‐9(Z),11(E)‐octadecadienoic acid into an unstable fatty acid allene oxide, 12,13(S)‐epoxy‐9(Z),11‐octadecadienoic acid. This study is concerned with the chemistry of 12,13(S)‐epoxy‐9(Z),11‐octadecadienoic acid in the presence of vertebrate serum albumins.Albumins were found to greatly enhance the aqueous half‐life of the allene oxide, i.e. 14.1±1.8 min, 11.6±1.2 min and 4.8±0.5 min at 0 C in the presence of 15 mg/ml of bovine, human and equine serum albumins, respectively, as compared with ca. 33 sec in the absence of albumin. Degradation of allene oxide in the presence of bovine serum albumin led to the formation of a novel cyclization product, i.e. 3‐oxo‐2‐pentyl‐cyclopent‐4‐en‐1‐octanoic acid (12‐oxo‐10‐phytoenoic acid, in which the relative configuration of the side chains attached to the five‐membered ring istrans). Steric analysis of the cyclic derivative showed that the compound was largely racemic (ratio between enantiomers, 58∶42).12‐Oxo‐10,15(Z)‐phytodienoic acid, needed for reference purposes, was prepared by incubation of 13(S)‐hydroperoxy‐9(Z),11(E),15(Z)‐octadecatrienoic acid with corn hydroperoxide dehydrase. Steric analysis showed that the 12‐oxo‐10,15(Z)‐phytodienoic acid thus obtained was not optically pure but a mixture of enantiomers in a ratio of 82∶18.

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.

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 alpha-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.