Lipoxygenase-mediated metabolism of storage lipids in germinating sunflower cotyledons and beta-oxidation of (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid by the cotyledonary glyoxysomes

During the early stages of germination, a lipid-body lipoxygenase is expressed in the cotyledons of sunflowers (Helianthus annuus L.). In order to obtain evidence for the in vivo activity of this enzyme during germination, we analyzed the lipoxygenase-dependent metabolism of polyunsaturated fatty acids esterified in the storage lipids. For this purpose, lipid bodies were isolated from etiolated sunflower cotyledons at different stages of germination, and the storage triacylglycerols were analyzed for oxygenated derivatives. During the time course of germination the amount of oxygenated storage lipids was strongly augmented, and we detected triacylglycerols containing one, two or three residues of (9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid. Glyoxysomes from etiolated sunflower cotyledons converted (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid to (9Z,11E)-13-oxo-octadeca-9,11-dienoic acid via an NADH-dependent dehydrogenase reaction. Both oxygenated fatty acid derivatives were activated to the corresponding CoA esters and subsequently metabolized to compounds of shorter chain length. Cofactor requirement and formation of acetyl-CoA indicate degradation via -oxidation. However, -oxidation only proceeded for two consecutive cycles, leading to accumulation of a medium-chain metabolite carrying an oxo group at C-9, equivalent to C-13 of the parent (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid. Short-chain -oxidation intermediates were not detected during incubation. Similar results were obtained when 13-hydroxy octadecanoic acid was used as -oxidation substrate. On the other hand, the degradation of (9Z,11E)-octadeca-9,11-dienoic acid was accompanied by the appearance of short-chain -oxidation intermediates in the reaction mixture. The results suggest that the hydroxyl/oxo group at C-13 of lipoxygenase-derived fatty acids forms a barrier to continuous -oxidation by glyoxysomes.     

Cationic substrates of soybean lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Synthesis of (S)-13-Hydroxy (2E,4E,8E)- and (2E,4E,8Z)-Tetradecatrienoic Acids

The syntheses of (S)-13-hydroxy-(2E,4E,8E)-tetradecatrienoic acid (1) and (2E,4E,8Z)-tetradecatrienoic acid (2) were carried out by using the Wittig reaction as the key step. The asymmetric center at C-13 and the double bond between C-8 and C-9 for natural compound 1 were reconfirmed as being of (S) configuration and E, respectively.

The relationship between the structure of the unsaturated hydroxy fatty acids and their inhibitory effect on the growth of lettuce was investigated.     

Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal : A Route to 4S-Hydroperoxy-2E-Nonenal and Related Products

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O₂ uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.     

Payne rearrangement during analysis of epoxyalcohols of linoleic and alpha-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry

Hydroperoxides of polyunsaturated fatty acids can be transformed to epoxyalcohols and keto fatty acids by metal enzymes, hematin, and various catalysts. In the current study, we used hematin to transform 9-hydroperoxy-10E,12Z-octadecadienoic acid and 13-hydroperoxy-9Z,11E-octadecadienoic acid to epoxyalcohols (with trans epoxide configuration) and to keto fatty acids. The products were separated by normal phase high-performance liquid chromatography (NP-HPLC) and analyzed using postcolumn addition of isopropanol/water and online negative ion electrospray ionization mass spectrometry (MS). The tandem MS (MS/MS) spectra were studied using analogs prepared from [9,10,12,13-²H₄]linoleic acid (18:2n−6) and from α-linolenic acid (18:3n−3). We also studied the MS/MS spectra of epoxyalcohols formed from 11-hydroperoxy- and 8-hydroperoxy-9Z,12Z-octadecadienoic acids. Results were confirmed by MS/MS analysis of a series of authentic standards. MS/MS ions of 9-keto-10E,12Z-octadecadienoic acid and 13-keto-9Z,11E-octadecadienoic acid could be explained by keto-enol tautomerism. MS/MS spectra of regioisomeric allylic epoxyalcohols differed in relative intensities of characteristic ions. The MS/MS spectra of the epoxyalcohols with 1-hydroxy-2,3-epoxy-4Z-pentene or 3-hydroxy-1,2-epoxy-4Z-pentene elements were virtually identical and showed two characteristic ions that differed by 30 in m/z values (CH(OH)). The results suggested that epoxide migration (Payne rearrangement) occurred during collision-induced dissociation. We conclude that regioisomeric allylic epoxyalcohols can be identified by their MS/MS spectra, whereas regioisomeric epoxyalcohols can be identified by MS/MS in combination with their retention times on NP-HPLC.     

E versus Z geometry in β-d-arabino-hexopyranosidulose oximes

Koenigs–Knorr-type glycosidations of peracylated 2Z-benzoyloxyimino-glycopyranosyl bromides invariably proceed with retention of the Z-geometry. Accordingly, the many β-d-hexosidulose oximes in literature which were prepared in this way and for which the oxime geometry has not been addressed explicitly, are the Z-oximes throughout. By contrast, oximation of β-d-hexopyranosid-2-uloses leads to mixtures of E and Z oximes readily separable and structurally verifiable by ¹H and ¹³C NMR. Configurational assignments rested on comparative evaluation of NMR data of E and Z isomers, and, most notably on an X-ray structural analysis of the pivaloylated isopropyl 2E-benzoyloxyimino-2-deoxy-β-d-arabino-hexopyranoside revealing the unusual ¹S₅^1,4B conformation for the pyranoid ring.     

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases

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

Geometrical Configuration of 12,13-Epoxyoctadeca-9,11-dienoic Acid,a Product of the Reaction Catalyzed by Flaxseed Allene Oxide Synthase (CYP74A)

The geometrical configuration of a short-living allene oxide reaction product that arises under the catalysis by flaxseed allene oxide synthase (CYP74A) was studied by NMR spectroscopy. The structure of (9Z, 11E)-12,13-epoxyoctadeca-9,11-dienoic acid was established for it from the results of the nuclear Overhauser effect measurements.     

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.     

Isomer-specific effects of conjugated linoleic acid on gene expression in RAW 264.7

Conjugated linoleic acid (CLA) is a mixture of dietary fatty acids that has various beneficial effects including decreasing cancer, atherosclerosis, diabetes and inflammation in animal models. Some controversy exists on the specific isomers of CLA that are responsible for the benefits observed. This study was conducted to examine how different CLA isomers regulate gene expression in RAW 264.7. A mouse macrophage cell line, RAW 264.7, was treated with five different CLA isomers (9E,11E-, 9Z,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA). Gene expression microarrays were performed, and several significantly regulated genes of interest were verified by a real-time polymerase chain reaction (PCR). Examination of the biological functions of various significantly regulated genes by the five CLA isomers showed distinct properties. Isomers 9E,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA decreased production of proinflammatory cytokines such as interleukin (IL)-1α, IL-1β and IL-6. Many of CLA's effects are believed to be mediated by the fatty acid receptors such as the peroxisome proliferator-activated receptors (PPAR) and retinoid-X-receptors (RXR). Using PPAR and RXR specific antagonists and coactivator recruitment assays, it was evident that multiple mechanisms were responsible for gene regulation by CLA isomers. Coactivator recruitment by CLA isomers showed their distinct properties as selective receptor modulators for PPARγ and RXRα. These studies demonstrate distinct isomer differences in gene expression by CLA and will have important ramifications for determining the potential therapeutic benefit of these dietary fatty acids in prevention of inflammation-related diseases.     

Biotransformation of sesquiterpenoids having α,β-unsaturated carbonyl groups with cultured plant cells of Marchantia polymorpha

The biotransformation of sesquiterpenoids having an α,β-unsaturated carbonyl group, such as α-santonin (1), lancerodiol p-hydroxybenzoate (2), 8,9-dehydronootkatone (3), and nootkatone (4), with cultured suspension cells of Marchantia polymorpha was investigated. It was found that the CC double bond of 1 and 2 was hydrogenated to give 1,2-dihydro-α-santonin (5) and 3,4-dihydrolancerodiol p-hydroxybenzoate (6), respectively, while the allylic position of the CC double bond of 3 and 4 was hydroxylated to give 13-hydroxy-8,9-dehydronootkatone (7) and 9-hydroxynootkatone (8), respectively.     

Isolation and spectral characterization of thermally generated multi-Z-isomers of lycopene and the theoretically preferred pathway to di-Z-isomers

Lycopene has a large number of geometric isomers caused by E/Z isomerization at arbitrary sites within the 11 conjugated double bonds, offering varying characteristics related to features such as antioxidant capacity and bioavailability. However, the geometric structures of only a few lycopene Z-isomers have been thoroughly identified from natural sources. In this study, seven multi-Z-isomers of lycopene, (9Z,13′Z)-, (5Z,13Z,9′Z)-, (9Z,9′Z)-, (5Z,13′Z)-, (5Z,9′Z)-, (5Z,9Z,5′Z)-, and (5Z,9Z)-lycopene, were obtained from tomato samples by thermal isomerization, and then isolated by elaborate chromatography, and fully assigned using proton nuclear magnetic resonance. Moreover, the theoretically preferred pathway from (all-E)-lycopene to di-Z-isomers was examined with a computational approach using a Gaussian program. Fine-tuning of the HPLC separation conditions led to the discovery of novel multi-Z-isomers, and whose formation was supported by advanced theoretical calculations.     

DHA Metabolism: Targeting the Brain and Lipoxygenation

Docosahexaenoic acid (DHA), the end-product of the metabolism of omega-3 family fatty acids, is the main polyunsaturated fatty acid of the brain, but its accumulation is incompletely understood. This paper reviews how it could accumulate through specific uptake of DHA-containing lysophosphatidylcholine (LysoPC-DHA). DHA migrates very easily from the sn-2 position of LysoPC, which could be considered as the physiological form of polyunsaturated LysoPC, to the sn-1 position, which is much more stable. An approach preventing migration by acetylating the sn-1 position, while retaining the main physico-chemical properties of the carrier, is described. Also, the double lipoxygenation and bond-isomerization of DHA into 10(S),17(S)-docosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid, named PDX, by soybean lipoxygenase is described. As in other E,Z,E conjugated trienes, PDX is shown to inhibit human blood platelet aggregation at submicromolar concentrations.     

Zeolites are effective ROS-scavengers in vitro

We report on the use of zeolites to limit the effects of reactive oxygen species (ROS) on human albumin under in vitro conditions. Zeolites of different structure type, channel size, channel polarity, and charge-compensating cation were screened for the elimination of ROS, notably HO, resulting from the Fenton reaction. A test based on ischemia-modified albumin (IMA) was used as a marker to monitor the activity of HO after co-exposure of human serum to these zeolites. Two commercial zeolites, faujasite (FAU 13×, channel opening 0.74 × 0.74 nm with Na⁺ as charge-compensating cation) and ferrierite (FER, channel opening 0.54 × 0.42 nm with H⁺ as charge-compensating cation), were found to reduce IMA formation by more than 65% due to removal of HO relative to reference values. It was established that partial ion exchange of the zeolites’ respective charge-compensating cation vs. Fe³⁺ implicated in the Fenton reaction plays a major role in HO deactivation process. Moreover, our results show that no saturation of the respective zeolite active sites occurred. This is possible only when ROS are actively converted to water molecules within the zeolite void system, which generates H⁺ ion transport.Because zeolites cannot be administered in blood, their use in medicine should be limited to extra corporeal circuits. Zeolites could be of use during cardiopulmonary bypass or hemodialysis procedures.     

Secretion of two novel enzymes,manganese 9S-lipoxygenase and epoxy alcohol synthase,by the rice pathogen Magnaporthe salvinii

The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion.     

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of ∼50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-²H]18:1n-9, [7R-²H]18:2n-6, and [8R-²H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.