Fatty Acid Hydroperoxides Research Articles

The hydroperoxide moiety of aliphatic lipid hydroperoxides is not affected by hypochlorous acid

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-​hydroperoxy-​5Z,​8Z,​11Z,​13E-​eicosatetraenoic acid (15-HpETE) and 13S-​hydroperoxy-​9Z,​11E-​octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

A high throughput screen identifies potent and selective inhibitors to human epithelial 15-lipoxygenase-2.

Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15­lipoxygenase­2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15­LOX­2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15­LOX­2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/−0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/−0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

Inhibitory effects of a novel Val to Thr mutation on the distal heme of human catalase.

True catalases efficiently breakdown hydrogen peroxide, whereas the catalase-related enzyme allene oxide synthase (cAOS) is completely unreactive and instead metabolizes a fatty acid hydroperoxide. In cAOS a Thr residue adjacent to the distal His restrains reaction with H2O2 (Tosha et al. (2006) J. Biol. Chem. 281:12610; De Luna et al. (2013) J. Phys. Chem. B 117: 14635) and its mutation to the consensus Val of true catalases permits the interaction. Here we investigated the effects of the reciprocal experiment in which the Val74 of human catalase is mutated to Thr, Ser, Met, Pro, or Ala. The Val74Thr substitution decreased catalatic activity by 3.5-fold and peroxidatic activity by 3-fold. Substitution with Ser had similar negative effects (5- and 3-fold decreases). Met decreased catalatic activity 2-fold and eliminated peroxidatic activity altogether, whereas the Val74Ala substitution was well tolerated. (The Val74Pro protein lacked heme). We conclude that the conserved Val74 of true catalases helps optimize catalysis. There are rare substitutions of Val74 with Ala, Met, or Pro, but not with Ser of Thr, possibly due their hydrogen bonding affecting the conformation of His75, the essential distal heme residue for activity in catalases.

Exposure assessment of Malondialdehyde, 4-Hydroxy-2-(E)-Nonenal and 4-Hydroxy-2-(E)-Hexenal through specific foods available in Belgium

Malondialdehyde (MDA), 4-Hydroxy-2-(E)-Nonenal (HNE) and 4-Hydroxy-2-(E)-Hexenal (HHE) are reactive aldehydes found in foods and are formed due to decomposition of polyunsaturated fatty acid hydroperoxides. In the present study, sixteen food categories were analyzed for the aforementioned aldehydes and in combination with consumption data obtained from a national representative sample of the Belgian population, a quantitative exposure assessment was performed. MDA was detected above the detection limit in 84% of the analyzed samples while HNE and HHE in 63% and 16% of the samples respectively. Consumption of dry nuts, fried snacks, French fries and cured minced meat products were found to contribute the most to the intake of MDA and HNE. Intake of HHE from the foods analyzed was found not to be significant. An evaluation of any potential risk related to the intake of the studied aldehydes through the studied foods was performed by applying the threshold of toxicological concern concept. No risk to human health could be identified related to the consumption of these foods for the vast majority of the consumers, with the only exception of a small proportion (3.8%) of those who consume cured and minced raw meat, that could be at risk.

Prevalence and mechanism of polyunsaturated aldehydes production in the green tide forming macroalgal genus Ulva (Ulvales, Chlorophyta)

Lipoxygenase/hydroperoxide lyase mediated transformations convert polyunsaturated fatty acids into various oxylipins. First, lipoxygenases catalyze fatty acid oxidation to fatty acid hydroperoxides. Subsequently, breakdown reactions result in a wide array of metabolites with multiple physiological and ecological functions. These fatty acid transformations are highly diverse in marine algae and play a crucial rule in e.g., signaling, chemical defense, and stress response often mediated through polyunsaturated aldehydes (PUAs). In this study, green tide-forming macroalgae of the genius Ulva (Chlorophyta) were collected at various sampling sites in the lagoon of the Ria Formosa (Portugal) and were surveyed for PUAs. We demonstrated that sea-lettuce like but not tube-like morphotypes produce elevated amounts of volatile C10-polyunsaturated aldehydes (2,4,7-decatrienal and 2,4-decadienal) upon tissue damage. Moreover, morphogenetic and phylogenetic analyses of the collected Ulva species revealed chemotaxonomic significance of the perspective biosynthetic pathways. The aldehydes are derived from omega-3 and omega-6 polyunsaturated fatty acids (PUFA) with 20 or 18 carbon atoms including eicosapentaenoic acid (C20:5 n-3), arachidonic acid (C20:4 n-6), stearidonic acid (C18:4 n-3), and γ-linolenic acid (C18:3 n-6). We present first evidences that lipoxygenase-mediated (11-LOX and 9-LOX) eicosanoid and octadecanoid pathways catalyze the transformation of C20- and C18-polyunsaturated fatty acids into PUAs and concomitantly into short chain hydroxylated fatty acids.

An Ancient Relative of Cyclooxygenase in Cyanobacteria Is a Linoleate 10S-Dioxygenase That Works in Tandem with a Catalase-related Protein with Specific 10S-Hydroperoxide Lyase Activity

In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as "cyclooxygenase-2," appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.

Sequence variation and haplotypes of lipoxygenase gene LOX-1 in the Australian barley varieties

BackgroundLipoxygenases are a family of enzymes which catalyse the hydroperoxidation of polyunsaturated fatty acids with a cis, cis-1,4-pentadiene to form conjugated hydroperoxydienes. Lipoxygenase-1 (LOX-1) in barley worsens the flavour and foam stability of beer. It has become a major selection criteria for malting quality in the last few years.ResultsLipoxygenase activity was investigated in 41 Australian barley cultivars and advanced breeding lines released since the 1950s; the cultivars differed markedly, ranging from 22.3 to 46.5 U/g. The structural gene and its promoter of lipoxygenase-1 were sequenced from the barley varieties representing different levels of LOX. Based on the analysis of nucleotide and deduced amino acid sequences, two major haplotypes were identified. Barley varieties with lower LOX were classified into three categories based on their pedigrees and sequence variations in the structural gene: (1) barley varieties derived from Canadian varieties with the pre-harvest sprouting susceptible allele, (2) Skiff and Hindmarsh with unique haplotype in the structural gene, and (3) Gairdner and Onslow with an unknown mechanism.ConclusionLipoxygenase activity has been reduced in the malting barley cultivars in the last 60 years although it is only recognized as a malting quality trait recently. There are clear haplotypes of the lipoxygenase structual gene. The polymorphisms detected in the structural gene can be used to design molecular markers for selection of low LOX haplotype. Other mechanisms also existed for controlling lipoxygenase activity. The results suggest that it is possible to develop barley varieties with lower LOX by combination of low LOX-1 haplotype and other trans-regulation factors.

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.

Prostate Epithelium-Specific Deletion of the Selenocysteine tRNA Gene Trsp Leads to Early Onset Intraepithelial Neoplasia

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.

A Molecular Dynamics Examination on Mutation-Induced Catalase Activity in Coral Allene Oxide Synthase

Coral allene oxide synthase (cAOS) catalyzes the formation of allene oxides from fatty acid hydroperoxides. Interestingly, its active site differs from that of catalase by only a single residue yet is incapable of catalase activity. That is, it is unable to catalyze the decomposition of hydrogen peroxide to molecular oxygen and water. However, the single active-site mutation T66V allows cAOS to exhibit catalase activity. We have performed a series of molecular dynamics (MD) simulations in order to gain insights into the differences in substrate (8R-hydroperoxyeicosatetraenoic) and H2O2 active site binding between wild-type cAOS and the T66V mutant cAOS. It is observed that in wild-type cAOS the active site Thr66 residue consistently forms a strong hydrogen-bonding interaction with H2O2 (catalase substrate) and, importantly, with the aid of His67 helps to pull H2O2 away from the heme Fe center. In contrast, in the T66V-cAOS mutant the H2O2 is much closer to the heme's Fe center and now forms a consistent Fe···O2H2 interaction. In addition, the His67···H2O2 distance shortens considerably, increasing the likelihood of a Cpd I intermediate and hence exhibiting catalase activity.

Reprogramming of fatty acid and oxylipin synthesis in rhizobacteria-induced systemic resistance in tomato

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion.

Traumatin- and Dinortraumatin-containing Galactolipids in Arabidopsis: THEIR FORMATION IN TISSUE-DISRUPTED LEAVES AS COUNTERPARTS OF GREEN LEAF VOLATILES

Green leaf volatiles (GLVs) consisting of six-carbon aldehydes, alcohols, and their esters, are biosynthesized through the action of fatty acid hydroperoxide lyase (HPL), which uses fatty acid hydroperoxides as substrates. GLVs form immediately after disruption of plant leaf tissues by herbivore attacks and mechanical wounding and play a role in defense against attackers that attempt to invade through the wounds. The fates and the physiological significance of the counterparts of the HPL reaction, the 12/10-carbon oxoacids that are formed from 18/16-carbon fatty acid 13-/11-hydroperoxides, respectively, are largely unknown. In this study, we detected monogalactosyl diacylglycerols (MGDGs) containing the 12/10-carbon HPL products in disrupted leaf tissues of Arabidopsis, cabbage, tobacco, tomato, and common bean. They were identified as an MGDG containing 12-oxo-9-hydroxy-(E)-10-dodecenoic acid and 10-oxo-7-hydroxy-(E)-8-decenoic acid and an MGDG containing two 12-oxo-9-hydroxy-(E)-10-dodecenoic acids as their acyl groups. Analyses of Arabidopsis mutants lacking HPL indicated that these MGDGs were formed enzymatically through an active HPL reaction. Thus, our results suggested that in disrupted leaf tissues, MGDG-hydroperoxides were cleaved by HPL to form volatile six-carbon aldehydes and non-volatile 12/10-carbon aldehyde-containing galactolipids. Based on these results, we propose a novel oxylipin pathway that does not require the lipase reaction to form GLVs.

Structure–function relationship in the CYP74 family: Conversion of divinyl ether synthases into allene oxide synthases by site-directed mutagenesis

Non-classical P450s of CYP74 family control several enzymatic conversions of fatty acid hydroperoxides to bioactive oxylipins in plants, some invertebrates and bacteria. The family includes two dehydrases, namely allene oxide synthase (AOS) and divinyl ether synthase (DES), and two isomerases, hydroperoxide lyase (HPL) and epoxyalcohol synthase. To study the interconversion of different CYP74 enzymes, we prepared the mutant forms V379F and E292G of tobacco (CYP74D3) and flax (CYP74B16) divinyl ether synthases (DESs), respectively. In contrast to the wild type (WT) enzymes, both mutant forms lacked DES activity. Instead, they produced the typical AOS products, α-ketols and (in the case of the flax DES mutant) 12-oxo-10,15-phytodienoic acid. This is the first demonstration of DES into AOS conversions caused by single point mutations.

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3: STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at -15 °C. One matched previously described allene oxides in its UV spectrum (λmax 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λmax 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.

Microwave Irradiation for Dry-Roasting of Hazelnuts and Evaluation of Microwave Treatment on Hazelnuts Peeling and Fatty Acid Oxidation

<p>European hazelnut is an important nut crop in Italy, where about 121,750 tons of in-shell nuts are produced every year. Roasting is the most important practice for hazelnut preservation and commonly is carried out in commercial electrical ovens at 120-160°C for 10-20 min. This needful practice is time and energy expensive, so the development of new processing methods is required to reduce processing costs and to obtain top quality roasted nuts. The aim of this study was to develop a simple microwave treatment for hazelnuts peeling and roasting.</p> <p>With this aim, some physical (colour, temperature, moisture) and chemical (taste, lipoxygenase activity, fatty acids, vitamins, sensory attributes) features of inshell nuts and kernels of three Italian hazelnut varieties (Tonda di Giffoni, Tonda Romana and Nocchione) after conventional oven or microwave roasting were evaluated.</p> <p>Results showed that microwave roasting of kernels for 450 s gave a higher peeling score than the conventional oven treatment. This paralleled with better colour and taste scores for microwaved roasted kernels. Furthermore, a 360-450 s microwave roasting was able to inactivate almost completely lipoxygenases, avoiding adverse effects on fatty acids hydroperoxides and PUFA content. A shorter microwave treatment (360 s) was enough to obtain good peeling and sensory scores of inshell hazelnuts.</p> Taken together our results indicated that microwave technology can be successfully applied to both kernels and inshell hazelnuts to obtain suitable peeling and high quality roasted nuts.

Resveratrol Biosynthesis Up-Regulates the Ascorbate/Glutathione Pathway in Transgenic Tomato Fruit

There is a growing interest in producing food plants with increased amounts of antioxidants because of their potential health benefits. Polyphenolic secondary metabolites, such as flavonoids and stilbenes, have been investigated for their significant antioxidant activity, important both for plant physiology and human nutrition. With the aim to verify whether the synthesis of resveratrol in tomato fruit affected the redox status of transformed tomato tissues, in the present work we took advantage of the availability of two different transgenic tomato lines in which a grape stilbene synthase gene was expressed under a constitutive or a tissue specific promoter, respectively. The induction of resveratrol synthesis in tomato affected the redox status of transformed tomato fruit. In particular, ascorbate and glutathione pool increased, significantly and proportionally to the amount of the resveratrol synthesised in transformed tissues. Noteworthy, the increase in ascorbate and glutathione pool paralleled with a significant increase in the activities of the main enzymes involved in redox homeostasis, i.e. catalase, ascorbate peroxidase as well the enzymes responsive for ascorbate recycling. Furthermore, lipoxygenase activity and levels of polyunsaturated fatty acids hydroperoxides, were reduced in fruit tissues from transgenic tomato lines. The synthesis of resveratrol, together with the global redox status had an impact on the total antioxidant activity of transgenic fruits. Again the total antioxidant capability increased proportionally to the amount of synthesised resveratrol with the most remarkable increase recorded from the lipophilic-resveratrol containing-fraction of transgenic fruits. Overall, our results pointed a higher nutritional value of resveratrol synthesizing tomato fruits.

Synthesis of aroma compounds of virgin olive oil: Significance of the cleavage of polyunsaturated fatty acid hydroperoxides during the oil extraction process

Synthesis of the aroma compounds of virgin olive oil (VOO) occurs through the lipoxygenase (LOX) pathway comprising mainly the actuation of LOX and hydroperoxide lyase (HPL) enzymes. The aim of this work was to determine whether the cleavage of polyunsaturated fatty acid hydroperoxides catalyzed by HPL is a limiting factor for the biosynthesis of VOO volatile compounds during the oil extraction process. For this purpose, HPL activity and the availability of substrates for this activity were modified during the oil extraction process from olive fruits of cultivars Arbequina and Picual, which give rise to oils with quantitatively different volatile profiles. Experimental data suggest that the HPL enzyme activity is just slightly limited during the oil extraction process in both cultivars, being this limitation apparently more significant during the processing of Arbequina fruits than of Picual fruits. However, this difference in HPL limitation seems to be more related to the differences in the amount of hydroperoxides produced in each cultivar than to the level of HPL activity during the olive processing.

How Plants Shut out Bacteria

Unlike animals, which breathe through airways lined with pathogen-trapping defenses, plants get air through tiny pores in their leaves that all but invite bacteria to sneak in. How, then, do plants keep them out? They slam their pores, or stomata, shut. Stomata are flanked by guard cells that swell when triggered by bacteria, thus closing the pores. Besides being fascinating in its own right, this defense response has implications for human health—stomata have recently been shown to block some, but not all, types of the fecal bacteria that can contaminate leafy greens and other fresh produce, causing food poisoning outbreaks. In this issue of PLOS Biology, Montillet, Leonhardt, and colleagues show that guard cells contain an enzyme called lipoxygenase that makes stomata close in response to pathogens, overturning a previous theory that this process is regulated by the plant hormone abscisic acid. Lipoxygenases have previously been linked to pathogen defenses in the plant Arabidopsis thaliana, and in the current work the researchers identified a lipoxygenase gene (LOX1) that is expressed in guard cells. To explore this gene's role in plant defenses, they sprayed LOX1-deficient A. thaliana with a strain of the bacterium Pseudomonas syringae that normally makes stomata close. However, they found that stomata barely closed in these mutant plants, showing that LOX1 is required for this defense against bacteria. Bolstering this conclusion, Montillet and colleagues also found that substrates and products of the LOX1 enzyme make stomata close. Lipoxygenases turn fatty acids into fatty acid hydroperoxides (FAHs), and turn FAHs into reactive electrophile species (RES) oxylipins. The researchers found that stomata closed in response to two fatty acids that are LOX1 substrates, as well as in response to LOX1-produced FAHs and RES oxylipins. Next, the researchers clarified the relative roles of LOX1 and abscisic acid (ABA), the plant hormone previously thought to mediate stomatal closure in this defense response. This set of experiments, which involved treating A. thaliana with a peptide from bacterial flagella that is known to activate plant defenses, yielded several lines of evidence that LOX1 and ABA activate guard cells via different pathways. For example, each pathway had two mitogen-activated protein kinases that were not in the other pathway. In addition, LOX1-produced RES oxylipins failed to activate OST1, a protein kinase in the ABA pathway. In contrast, RES oxylipins made stomata close in A. thaliana mutants that either lacked OST1 or could not synthesize ABA. Finally, stomata close within an hour of the flagellar peptide treatment, but ABA marker genes were not expressed, suggesting that ABA was not produced during this time period. Moreover, Montillet and colleagues discovered that only the LOX pathway includes another plant hormone called salicylic acid, which is required for stomatal closure in response to bacteria. They found that the RES oxylipins produced by LOX1 failed to activate guard cells in A. thaliana mutants that lacked salicylic acid. In contrast, ABA still activated guard cells in these salicylic acid–deficient mutants. Besides suggesting that ABA and LOX1 are in different pathways, this work suggests that the LOX1 pathway makes plants synthesize salicylic acid, and that this is a key step in activating guard cells to shut out bacteria. So what does trigger the ABA pathway? Guard cells also close in response to environmental conditions such as light and humidity, suggesting that ABA mediates stomatal closure in response to such abiotic factors rather than to biological ones. While the researchers conclude that the ABA and LOX1 pathways are distinct and have different triggers, they also stress that both are required for stomata to close in response to bacteria. This is because regardless of whether the trigger is biotic or abiotic, ABA is the main regulator of the membrane proteins that control osmotic pressure in guard cells, which is what determines whether stomata are open or shut. Notably, an ion channel called SLAC1 is required for both biotic and abiotic stomatal closure, suggesting that both the ABA and LOX1 pathways act on this membrane protein. These findings extend previous work showing that stomata are key players in the defense response of plants, and significantly advance our understanding of stomatal closure. This could ultimately help us protect people from food plants that can carry human pathogens. In addition, because stomata regulate water loss, teasing apart the abiotic and biotic pathways of stomatal closure could help us breed plants better suited to harsh environmental conditions. Montillet J-L, Leonhardt N, Mondy S, Tranchimand S, Rumeau D, et al. (2013) An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis. doi:10.1371/journal.pbio.1001513

Plasma HDL Reduces Nonesterified Fatty Acid Hydroperoxides Originating from Oxidized LDL: a Mechanism for Its Antioxidant Ability

The antioxidant property of plasma high-density lipoprotein (HDL) is thought to be involved in potential anti-atherogenic effects but the exact mechanism is not known. We aimed to reveal the contribution of HDL on the elimination of lipid hydroperoxides (LOOH) derived from oxidized low-density lipoprotein (LDL). Oxidized LDL prepared by copper ion-induced oxidation contained nonesterified fatty acid hydroperoxides (FFA-OOH) and lysophosphatidylcholine (lysoPtdCho), in addition to cholesteryl ester hydroperoxides (CE-OOH) and phosphatidylcholine hydroperoxides (PtdCho-OOH). A platelet-activating factor-acetylhydrolase (PAF-AH) inhibitor suppressed formation of FFA-OOH and lysoPtdCho in oxidized LDL. Among LOOH species, FFA-OOH was preferentially reduced by incubating oxidized LDL with HDL. HDL exhibited selective FFA-OOH reducing ability if it was mixed with a liposomal solution containing FFA-OOH, CE-OOH and PtdCho-OOH. Two-electron reduction of the hydroperoxy group to the hydroxy group was confirmed by the formation of 13-hydroxyoctadecadienoic acid from 13-hydroperoxyoctadecadienoic acid in HPLC analyses. This reducing effect was also found in apolipoprotein A-1 (apoA-1). FFA-OOH released from PtdCho-OOH due to PAF-AH activity in oxidized LDL undergo two-electron reduction by the reducing ability of apoA1 in HDL. This preferential reduction of FFA-OOH may participate in the mechanism of the antioxidant property of HDL.

Biosynthesis of allene oxides in Physcomitrella patens

BackgroundThe moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2.ResultsOur results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS – a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis.ConclusionsOur study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.