Fatty Acid Hydroperoxides Research Articles

Engineering the substrate specificity and regioselectivity of Burkholderia thailandensis lipoxygenase

Lipoxygenases (LOXs) catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), generating fatty acid hydroperoxides (FAHPs) with diverse industrial applications. Bacterial LOXs have garnered significant attention in recent years due to their broad activity towards PUFAs, yet knowledge about the structural factors influencing their substrate preferences remains limited. Here, we characterized a bacterial LOX from Burkholderia thailandensis (Bt-LOX), and identified key residues affecting its substrate preference and regioselectivity through site-directed mutagenesis. Bt-LOX preferred ω-6 PUFAs and exhibited regioselectivity at the ω-5 position. Mutations targeting the substrate binding pocket and the oxygen access channel led to the production of three active variants with distinct catalytic properties. The A431G variant bifurcated dioxygenation between the ω-5 and ω-9 positions, while F446V showed reduced regioselectivity with longer PUFAs. Interestingly, L445A displayed altered substrate specificity, favoring ω-3 over ω-6 PUFAs. Furthermore, L445A shifted the regioselectivity of dioxygenation to the ω-2 position in ω-3 PUFAs, and, for some substrates, facilitated dioxygenation closer to the carboxylic acid terminus, suggesting an altered substrate orientation. Among these variants, L445A represents a significant milestone in LOX research, as these alterations in substrate specificity, dioxygenation regioselectivity, and substrate orientation were achieved by a single mutation only. These findings illuminate key residues governing substrate preference and regioselectivity in Bt-LOX, offering opportunities for synthesizing diverse FAHPs and highlighting the potential of bacterial LOXs as biocatalysts with widespread applications.

Heme-catalyzed degradation of linoleate 9-hydroperoxide (9-HPODE) forms two allylic epoxy-ketones via a proposed pseudo-symmetrical diepoxy radical intermediate

Heme-initiated decomposition of unsaturated fatty acid hydroperoxides creates alkoxyl radicals that propagate a complex series of reactions to hydroxy, keto, epoxy and aldehydic products. Herein, among the products from the hematin-catalyzed degradation of 9-hydroperoxy-linoleic acid (9-HPODE), we observed a double peak on normal-phase HPLC that resolved on RP-HPLC into equal proportions of two epoxy-allylic ketones with identical UV spectra. Their proton NMR spectra were also indistinguishable and consistent with 9,10-trans-epoxy-11E-13-keto- and 9-keto-10E-12,13-trans-epoxy-octadecenoic acids. Acid hydrolysis to the corresponding dihydroxy-ketones and GC-MS analysis identified the earlier eluting product on RP-HPLC as the 9,10-epoxy regio-isomer. Starting from the C9-hydroperoxide, recovery of the two epoxy-ketones in equal proportions suggests their formation from a common intermediate. Earlier work has proposed formation of a pseudo-symmetrical diepoxy radical (9,10-epoxy-11(•)-12,13-epoxy, derived from an epoxy allylic hydroperoxide precursor) in the carbon chain fragmentation leading to aldehydic products. This intermediate in pathways of alkoxyl radical reactions forms equal pairs of aldehydes, and now also a pair of epoxy-ketones, and based on mechanism the same products arise from either 9-HPODE or 13-HPODE. Our results point to the intermediacy of this diepoxy-carbinyl radical in the origin of at least two classes of linoleate peroxidation products, and it should be considered as a viable intermediate for homo-conjugated diene peroxidation in general. The reactions could contribute to the aldehydes and epoxy-ketones in tissues undergoing oxidative transformations of polyunsaturated fatty acids.

Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke.

Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two AOS genes HvnAOS1 and HvnAOS2 in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13(S)-HPOT, respectively. The expression pattern also indicated that HvnAOS1 and HvnAOS2 were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of HvnAOS1 and HvnAOS2 using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the cell cytoplasm of the N. benthamiana leaves. Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to Botrytis cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.

Versatile ferrous oxidation-xylenol orange assay for high-throughput screening of lipoxygenase activity.

Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as biocatalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under different conditions. This study aimed to enhance the suitability of the ferrous-oxidized xylenol orange (FOX) assay for screening LOX activity across a wide pH range with different PUFAs. The narrow linear detection range of the standard FOX assay restricts its utility in screening LOX activity. To address this, the concentration of perchloric acid in the xylenol orange reagent was adjusted. The modified assay exhibited a fivefold expansion in the linear detection range for hydroperoxides and accommodated samples with pH values ranging from 3 to 10. The assay could quantify various hydroperoxide species, indicating its applicability in assessing LOX substrate preferences. Due to sensitivity to pH, buffer types, and hydroperoxide species, the assay required calibration using the respective standard compound diluted in the same buffer as the measured sample. The use of correction factors is suggested when financial constraints limit the use of FAHP standard compounds in routine LOX substrate preference analysis. FAHP quantification by the modified FOX assay aligned well with results obtained using the commonly used conjugated diene method, while offering a quicker and broader sample pH range assessment. Thus, the modified FOX assay can be used as a reliable high-throughput screening method for determining LOX activity. KEY POINTS: • Modifying perchloric acid level in FOX reagent expands its linear detection range • The modified FOX assay is applicable for screening LOX activity in a wide pH range • The modified FOX assay effectively assesses substrate specificity of LOX.

Effects of fatty acid hydroperoxides produced by lipoxygenase in wheat cultivars during dough preparation on volatile compound formation

The formation of volatile compounds affects the flavor of processed wheat flour products. Herein, the effects of the composition of fatty acid hydroperoxides and the differences in the antioxidant contents among wheat cultivars on the flavor of wheat flour products were clarified. For this purpose, the volatile compounds in wheat flour doughs, LOX activity, fatty acid hydroperoxide composition from fractionated LOX, and antioxidant content were analyzed. Norin61 exhibited a high LOX activity and 9-fatty acid hydroperoxide production. Unsaturated aldehydes derived from 9-fatty acid hydroperoxides contributed significantly to the volatile compound profile of Norin61. Moreover, the lowest lutein content was observed in Norin61 among the analyzed cultivars. The LOX activity and composition of the fatty acid hydroperoxides produced by LOX affected the production of volatile compounds, whereas carotenoids had a suppressive effect. This study provides useful information for product design with the desired flavor for developing various processed wheat flour products.

Comparison of the Catabolic Rates of Linoleic and Oleic Acid Hydroperoxides Using 13CO2 Expired from Mice.

Unsaturated fatty acids, such as oleic and linoleic acids, are easily oxidized by exposure to temperature and light in the presence of air to form unsaturated fatty acid hydroperoxides as primary oxidation products. However, the catabolic rates of unsaturated fatty acid hydroperoxides in the human body remain unknown. In this study, ethyl esters of 13C-labeled linoleic acid (*C18:2-EE) and oleic acid (*C18:1-EE) and their hydroperoxides (*C18:2-EE-OOH and *C18:1-EE-OOH, respectively) prepared by the photo-oxidation of *C18:2-EE and *C18:1-EE, respectively, were administered to mice and their catabolic rates were determined by measuring the expired 13CO2 levels. *C18:2-EE-OOH and *C18:1-EE-OOH were β-oxidized faster than *C18:2-EE and *C18:1-EE, respectively. Notably, rapid β-oxidation of *C18:2-EE-OOH and *C18:1-EE-OOH was similar to that of medium-chain fatty acids, such as octanoic acid. Then, degradation products of C18:2-EE-OOH and C18:1-EE-OOH were analyzed under gastric conditions by gas chromatography/mass spectrometry. Major decomposition products of C18:2-EE-OOH and C18:1-EE-OOH were medium-chain compounds, such as octanoic acid ethyl ester, 9-oxo-nonanoic acid ethyl ester, and 10-oxo-8-decenoic acid ethyl esters, indicating that C18:2-EE-OOH and C18:1-EE-OOH isomers formed during photo-oxidation were decomposed under acidic conditions. These findings support previous reports that dietary lipid hydroperoxides are not absorbed into the intestine as lipid hydroperoxides but as degradation products. This is the first study to suggest that dietary lipid hydroperoxides decompose during gastric digestion to form medium-chain compounds that are directly absorbed into the liver via the portal vein and rapidly catabolized via β-oxidation.

Preparation of saturated fatty acid hydroperoxide isomers and determination of their thermal decomposition products – 2-alkanones and lactones

As known for quite a long time now, even saturated fatty acids can be oxidized at high temperatures to produce unique aroma compounds, such as 2-alkanones and lactones. Hydroperoxide positional isomers with a hydroperoxy group at the 2-, 3-, 4-, or 5-position are hypothesized to be responsible for the formation of these aroma components, but this hypothesis has not been verified. For the first time, this study successfully prepared a series of glyceryl trioctanoate hydroperoxide (C8TG;OOH) isomers. The isomers were thermally decomposed, proving that 2-heptanone was selectively formed from C8TG;3-OOH, and γ- and δ-octalactones were mainly formed from C8TG;4- and 5-OOH, respectively. C8TG;2-OOH was also involved in lactone formation, whereas C8TG;6- and 7-OOH were not. This proves the long-standing hypothesis. The mechanism revealed in this work is expected to be useful to create favorable aromas (i.e., 2-alkanones and lactones) from saturated fatty acids.

The beneficial effects of the tobacco hydroperoxide lyase pathway in whitefly host adaptation

Plants respond to herbivore attack by emitting complex mixtures of volatile compounds to repel herbivores or attract predators and parasitoids. In this study, however, we revealed that tobacco green leaf volatiles (GLVs) were beneficial compounds for whitefly adaptation to host plants. Our study indicates that GLVs are closely associated with plant susceptibility to whiteflies. Whitefly infestation elevated the transcript of the hydroperoxide lyase (HPL) gene, one gene responsible for catalyzing the synthesis of C6 or C9 aldehydes from fatty acid hydroperoxides. Overexpression of HPL increased the emission of GLVs (hexanal, 1- hexanal, trans-2- hexanal and cis-3- hexanal) and resulted in improved performance for whiteflies. Exogenous application of GLVs promoted whitefly survival and fecundity and increased the plant's attraction to whiteflies. Thus, our study provides new insights into the role of the HPL pathway and GLVs in influencing herbivore adaptation to host plants.

Molecular, in silico and expression studies on lipoxygenases (LOXs) in potato (Solanum tuberosum L.).

Lipoxygenases (LOXs) namely 9-LOXs and 13-LOXs catalyse the oxygenation of polyunsaturated fatty acids to produce fatty acid hydroperoxides which are crucial in growth, development and stress responses in plants. Here, we isolated and characterized a 2723-bp cDNA encoding a distinct 861-aa 9-LOX form, designated StKCLX-1, using tuber total RNA from an Indian potato cultivar, Kufri Chipsona-1 through RT-PCR. A total of 17 LOX genes distributed in different chromosomes were identified and characterized in the potato genome. Multiple sequence alignment revealed highly conserved amino acids in the crucial domains, motifs and variable N-terminal regions between the LOX classes. A total of 36 LOXs from potato, tomato and Arabidopsis were used in phylogenetic analysis. A 3-D structure of StKCLX-1 was predicted by AlphaFold tool, validated through the predicted local-distance difference test (pLDDT) and Ramachandran Plot. Molecular docking predicted the nature of receptor-ligand interactions. STRING database was used to predict the protein-protein interactions. Expression patterns of the LOXs in the potato organs were examined by Expression Atlas and semi-quantitative RT-PCR. 9-LOX activity was noticed at early stages of tuberization, and significantly increased in the freshly-harvested mature tubers. This report would be useful in gaining insights into the structure-function relationships of the LOXs and corresponding multigene family-prerequisites for understanding tuber development in potato.

Intramolecular H-Atom Transfers in Alkoxyl Radical Intermediates Underlie the Apparent Oxidation of Lipid Hydroperoxides by Fe(II).

The one-electron reduction of lipid hydroperoxides by low-valent iron species is believed to be a driver of cellular lipid peroxidation and associated ferroptotic cell death. We investigated reactions of cholesterol 7α-OOH, the primary cholesterol autoxidation product, with Fe2+ to find that 7-ketocholesterol (7-KC, an oxidation product) is the major product under these (reducing) conditions. Mechanistic studies reveal the intervention of a 1,2-H-atom shift upon formation of the 7-alkoxyl radical to yield a ketyl radical that can be oxidized by either Fe3+ or O2 to give 7-KC, the most abundant oxysterol in vivo. We also investigated the corresponding reduction of the isomeric cholesterol 5α-OOH and again found that an oxidation product (5-hydroxycholesten-3-one) predominates under reducing conditions. An intramolecular H-atom shift (this time 1,4-) in the initially formed 5-alkoxyl radical is suggested to yield a ketyl radical that is oxidized to give the observed product. It would appear that a 1,2-H shift also accounts for the predominance of ketones over alcohols when unsaturated fatty acid hydroperoxides are exposed to iron-based reductants, which had previously been reported with hematin and demonstrated here with Fe2+. The predominance of 7-KC over the corresponding alcohol is maintained when cholesterol 7α-OOH embedded in phospholipid liposomes is treated with Fe2+ or when ferroptosis is induced in mouse embryonic fibroblasts. Our observation that 7-KC accumulates in ferroptotic cells suggests that it may be a good biomarker for ferroptosis.

Advantages of hydroxyl radical water treatment for the fruits and vegetables preservation

In this study, the effects of hydroxyl radical water (HRW) treatment on the functional components, volatile compounds, and preservation properties of cherry tomatoes, grapes, coriander, and celery were investigated. The results demonstrated that HRW treatment could be significantly effective in delaying the color change and decay of fruits and vegetables. The loss of total sugars in the HRW-treated samples was relatively low, ranging from 0.02% to 0.84%. However, other functional components will be more severely affected. Specifically, celery exhibited a 17.95% decrease in total phenols and a 13.01% decrease in ascorbic acid, which may be caused by excessive surface area, indicating that the applicability of HRW to celery remains to be studied. The loss of functional components of both fruits could be controlled under 4%. Throughout the treatment, the phenolic profiles and volatile compounds remained stable. Notably, an increase in partial ester compounds, such as ethyl caproate, was observed in HRW-treated cherry tomatoes, which might improve the sensory function. Moreover, the reduction of some compounds obtained from the decomposition of fatty acid hydroperoxides may enhance the fresh odor of fruits and vegetables. Consequently, HRW treatment emerges as a safe and environmentally friendly technology for postharvest processing of fruits and vegetables.

Mitochondrial Peroxiredoxin 3 Is Rapidly Oxidized and Hyperoxidized by Fatty Acid Hydroperoxides.

Human peroxiredoxin 3 (HsPrx3) is a thiol-based peroxidase responsible for the reduction of most hydrogen peroxide and peroxynitrite formed in mitochondria. Mitochondrial disfunction can lead to membrane lipoperoxidation, resulting in the formation of lipid-bound fatty acid hydroperoxides (LFA-OOHs) which can be released to become free fatty acid hydroperoxides (fFA-OOHs). Herein, we report that HsPrx3 is oxidized and hyperoxidized by fFA-OOHs including those derived from arachidonic acid and eicosapentaenoic acid peroxidation at position 15 with remarkably high rate constants of oxidation (>3.5 × 107 M-1s-1) and hyperoxidation (~2 × 107 M-1s-1). The endoperoxide-hydroperoxide PGG2, an intermediate in prostanoid synthesis, oxidized HsPrx3 with a similar rate constant, but was less effective in causing hyperoxidation. Biophysical methodologies suggest that HsPrx3 can bind hydrophobic structures. Indeed, molecular dynamic simulations allowed the identification of a hydrophobic patch near the enzyme active site that can allocate the hydroperoxide group of fFA-OOHs in close proximity to the thiolate in the peroxidatic cysteine. Simulations performed using available and herein reported kinetic data indicate that HsPrx3 should be considered a main target for mitochondrial fFA-OOHs. Finally, kinetic simulation analysis support that mitochondrial fFA-OOHs formation fluxes in the range of nM/s are expected to contribute to HsPrx3 hyperoxidation, a modification that has been detected in vivo under physiological and pathological conditions.

From linoleic acid to hexanal and hexanol by whole cell catalysis with a lipoxygenase, hydroperoxide lyase and reductase cascade in Komagataella phaffii.

Green leaf volatiles (GLVs) cover a group of mainly C6-and C9-aldehydes, -alcohols and -esters. Their name refers to their characteristic herbal and fruity scent, which is similar to that of freshly cut grass or vegetables. Lipoxygenases (LOXs) catalyze the peroxidation of unsaturated fatty acids. The resulting hydroperoxy fatty acids are then cleaved into aldehydes and oxo acids by fatty acid hydroperoxide lyases (HPLs). Herein, we equipped the yeast Komagataella phaffii with recombinant genes coding for LOX and HPL, to serve as a biocatalyst for GLV production. We expressed the well-known 13S-specific LOX gene from Pleurotus sapidus and a compatible HPL gene from Medicago truncatula. In bioconversions, glycerol induced strains formed 12.9mM hexanal using whole cells, and 8mM hexanol was produced with whole cells induced by methanol. We applied various inducible and constitutive promoters in bidirectional systems to influence the final ratio of LOX and HPL proteins. By implementing these recombinant enzymes in Komagataella phaffii, challenges such as biocatalyst supply and lack of product specificity can finally be overcome.

Arachidonate 15-lipoxygenase type B: Regulation, function, and its role in pathophysiology.

As a lipoxygenase (LOX), arachidonate 15-lipoxygenase type B (ALOX15B) peroxidizes polyenoic fatty acids (PUFAs) including arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and linoleic acid (LA) to their corresponding fatty acid hydroperoxides. Distinctive to ALOX15B, fatty acid oxygenation occurs with positional specificity, catalyzed by the non-heme iron containing active site, and in addition to free PUFAs, membrane-esterified fatty acids serve as substrates for ALOX15B. Like other LOX enzymes, ALOX15B is linked to the formation of specialized pro-resolving lipid mediators (SPMs), and altered expression is apparent in various inflammatory diseases such as asthma, psoriasis, and atherosclerosis. In primary human macrophages, ALOX15B expression is associated with cellular cholesterol homeostasis and is induced by hypoxia. Like in inflammation, the role of ALOX15B in cancer is inconclusive. In prostate and breast carcinomas, ALOX15B is attributed a tumor-suppressive role, whereas in colorectal cancer, ALOX15B expression is associated with a poorer prognosis. As the biological function of ALOX15B remains an open question, this review aims to provide a comprehensive overview of the current state of research related to ALOX15B.

The Biosynthesis of 1-octene-3-ol by a Multifunctional Fatty Acid Dioxygenase and Hydroperoxide Lyase in Agaricus bisporus.

The biosynthetic pathway from linoleic acid to 1-octen-3-ol in Agaricus bisporus has long been established, in which linoleic acid is converted to 10-hydroperoxide (10-HPOD) by deoxygenation, and 10-HPOD is subsequently cleaved to yield 1-octene-3-ol and 10-oxodecanoic acid. However, the corresponding enzymes have not been identified and cloned. In the present study, four putative genes involved in oxylipid biosynthesis, including one lipoxygenase gene named AbLOX, two linoleate diol synthase genes named AbLDS1 and AbLDS2, and one hydroperoxide lyase gene named AbHPL were retrieved from the A. bisporus genome by a homology search and cloned and expressed prokaryotically. AbLOX, AbLDS1, and AbLDS2 all exhibited fatty acid dioxygenase activity, catalyzing the conversion of linoleic acid to generate hydroperoxide, and AbHPL showed a cleaving hydroperoxide activity, as was determined by the KI-starch method. AbLOX and AbHPL catalyzed linoleic acid to 1-octen-3-ol with an optimum temperature of 35 °C and an optimum pH of 7.2, whereas AbLDS1, AbLDS2, and AbHPL catalyzed linoleic acid without 1-octen-3-ol. Reduced AbLOX expression in antisense AbLOX transformants was correlated with a decrease in the yield of 1-octen-3-ol. AbLOX and AbHPL were highly homologous to the sesquiterpene synthase Cop4 of Coprinus cinerea and the yeast sterol C-22 desaturase, respectively. These results reveal that the enzymes for the oxidative cleavage of linoleic acid to synthesize 1-octen-3-ol in A. bisporus are the multifunctional fatty acid dioxygenase AbLOX and hydroperoxide lyase AbHPL.

Validation of a high-throughput method for the accurate quantification of secondary products of lipid oxidation in high-quality hazelnuts (Corylus avellana L.): A robust tool for quality assessment

European hazelnuts (Corylus avellana L.) are a key ingredient for the confectionery industry with a distinctive flavor profile and high nutritional quality. However, the high-fat content makes raw kernels prone to autoxidation that takes place during storage. Fatty acids hydroperoxides (i.e., primary products of oxidation) readily decompose by forming volatile secondary products with low odor thresholds and negative aroma notes. This study presents method performance parameters of an accurate quantitative methodology, based on multiple headspace solid-phase microextraction (MHS-SPME) coupled to conventional and fast gas chromatography–mass spectrometry ((Fast)GC–MS), targeted to oleic and linoleic acids autoxidation products (i.e., hexanal, heptanal, octanal, and nonanal). Results refer to good repeatability (max RSDr 12%) and intermediate precision (max RSDR 19.6%); linearity over four orders of magnitude (R2 ≥ 0.995 and calibration error ≤ 20%); good accuracy (error bias ≤20%); limit of detection and quantification compatible with real-world samples oxidative levels. Experimental results from method application on 358 industrial samples, confirm the method's effectiveness and its information capacity; moreover, for medium-to-high oxidized samples, the quantification of a larger set of products instead of a single marker (i.e., hexanal), provides a more accurate picture on the actual quality/rancidity level.

Biosynthesis of natural aroma compounds using recombinant whole-cell tomato hydroperoxide lyase biocatalyst

Green leaf volatiles impart characteristic aroma and flavour to a variety of natural foods due to their inherent grassy note contributed by aldehydes. Hydroperoxide lyase (HPL) is an enzyme that helps in the cleavage of fatty acid hydroperoxides to short-chain aldehydes and ω-oxo-acids. A tomato hydroperoxide lyase gene was successfully expressed in E. coli BL21 (DE3) cells and used in the subsequent production of (Z)-3-hexenal. Biochemical characterization of the HPL activity exhibited by these whole cells enabled the development of a suitable one-pot reaction process for conversion of the hydroperoxide substrate to the corresponding aldehyde, (Z)-3-hexenal, and finally to (Z)-3-hexenol, a high-value flavour and fragrance ingredient.

Reprint of: Photoperoxidation in Isolated Chloroplasts I. Kinetics and Stoichiometry of Fatty Acid Peroxidation

A photo-induced cyclic peroxidation in isolated chloroplasts is described. In an osmotic buffered medium, chloroplasts upon illumination produce malondialdehyde (MDA)—a decomposition product of tri-unsaturated fatty acid hydroperoxides—bleach endogenous chlorophyll, and consume oxygen. These processes show (a) no reaction in the absence of illumination; (b) an initial lag phase upon illumination of 10-20 minutes duration; (c) a linear phase in which the rate is proportional to the square root of the light intensity; (d) cessation of reaction occurring within 3 minutes after illumination ceases; and (e) a termination phase after several hours of illumination. The kinetics of the above processes fit a cyclic peroxidation equation with velocity coefficients near those for chemical peroxidation.The stoichiometry of MDA/O2 = 0.02, and O2/Chlbleached = 6.9 correlates well with MDA production efficiency in other biological systems and with the molar ratio of unsaturated fatty acids to chlorophyll. The energies of activation for the lag and linear phases are 17 and 0 kcal/mole, respectively, the same as that for autoxidation. During the linear phase of oxygen uptake the dependence upon temperature and O2 concentration indicates that during the reaction, oxygen tension at the site of peroxidation is 100-fold lower than in the aqueous phase.It is concluded that isolated chloroplasts upon illumination can undergo a cyclic peroxidation initiated by the light absorbed by chlorophyll. Photoperoxidation results in a destruction of the chlorophyll and tri-unsaturated fatty acids of the chloroplast membranes.

Detection of divinyl ether synthase CYP74H2 biosynthesizing (11Z)-etheroleic and (1ʹZ)-colnelenic acids in asparagus (Asparagus officinalis L.)

Divinyl ether synthases (DESs) are the enzymes occurring in numerous plant species and catalysing the dehydration of fatty acid hydroperoxides to divinyl ether oxylipins, playing self-defensive and antipathogenic roles in plants. Previously, the DES activities and divinyl ethers were detected in some monocotyledonous plants, including the asparagus (Asparagus officinalis L.). The cloning of the open reading frame of the CYP74H2 gene of asparagus and catalytic properties of the recombinant CYP74H2 protein are described in the present work. The CYP74H2 utilized the 13(S)-hydroperoxide of linoleic acid (13(S)-HPOD) as a preferred substrate and specifically converted it to the divinyl ether, (9Z,11Z)-12-[(1ʹE)-hexenyloxy]-9,11-dodecadienoic acid, (11Z)-etheroleic acid. The second most efficient substrate after the 13(S)-HPOD was the 9(S)-hydroperoxide of α−linolenic acid (9(S)-HPOT), which was converted to the previously undescribed product, (1ʹZ)-colnelenic acid. The 13(S)-hydroperoxide of α−linolenic acid (13(S)-HPOT) and 9(S)-hydroperoxide of linoleic acid (9(S)-HPOD) were less efficient substrates for CYP74H2. Both 13(S)-HPOT and 9(S)-HPOD were transformed to divinyl ethers, (11Z)-etherolenic and (1ʹZ)-colneleic acids, respectively. The CYP74H2 is a second cloned monocotyledonous DES after the garlic CYP74H1 and the first DES biosynthesizing the (1ʹZ)-colneleic and (1ʹZ)-colnelenic acids.

Determination of acrolein generation pathways from linoleic acid and linolenic acid: increment by photo irradiation

2-Propenal (acrolein) is a toxic aldehyde generated from the thermal degradation of edible oils. While previous studies have suggested that linolenic acid (LnA) is the origin of acrolein formation in edible oils, these studies were performed under thermal conditions where only the fatty acid hydroperoxide (FAOOH) isomers derived from radical oxidation were formed. In this study, we reinvestigated the acrolein generation pathway through another oxidation mechanism involving singlet oxygen (1O2) oxidation (type II photo-oxidation). Standards of the main FAOOH isomers (oleic acid hydroperoxide, linoleic acid hydroperoxide (HpODE), and linolenic acid hydroperoxide (HpOTE)) found in edible oils were prepared, and their decomposition products, including those derived from1O2 oxidation (i.e., 10- and 12-HpODE) were analyzed by GC-EI-MS. We found that 1O2 oxidation products of linoleic acid (LA) and LnA but not OA, are significant sources of acrolein formation. The amount of acrolein formed from edible oils high in LA (e.g., rice bran oil) increased by photo irradiation. Further investigation into the mechanism of acrolein generation demonstrated that the amount of acrolein derived from 1O2 oxidation-specific HpOTE isomers (i.e., 10- and 15-HpOTE) was two times greater than that of other HpOTE isomers (i.e., 9-, 12-, 13-, and 16-HpOTE). The results of the present study provide a new pathway of acrolein formation from type II photo-oxidation. This information can be used to inform on oil storage and processing conditions to reduce exposure and dietary intake of acrolein.