PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals.

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Impact of N-Glycosylation on Protein Structure and Dynamics Linked to Enzymatic C-H Activation in the M. oryzae Lipoxygenase.

Lipoxygenases (LOXs) from pathogenic fungi are potential therapeutic targets for defense against plant and select human diseases. In contrast to the canonical LOXs in plants and animals, fungal LOXs are unique in having appended N-linked glycans. Such important post-translational modifications (PTMs) endow proteins with altered structure, stability, and/or function. In this study, we present the structural and functional outcomes of removing or altering these surface carbohydrates on the LOX from the devastating rice blast fungus, M. oryzae, MoLOX. Alteration of the PTMs did notinfluence the active site enzyme-substrate ground state structures as visualized by electron-nuclear double resonance (ENDOR) spectroscopy. However, removal of the eight N-linked glycans by asparagine-to-glutamine mutagenesis nonetheless led to a change in substrate selectivity and an elevated activation energy for the reaction with substrate linoleic acid, as determined by kinetic measurements. Comparative hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of wild-type and Asn-to-Gln MoLOX variants revealed a regionally defined impact on the dynamics of the arched helix that covers the active site. Guided by these HDX results, a single glycan sequon knockout was generated at position 72, and its comparative substrate selectivity from kinetics nearly matched that of the Asn-to-Gln variant. The cumulative data from model glyco-enzyme MoLOX showcase how the presence, alteration, or removal of even a single N-linked glycan can influence the structural integrity and dynamics of the protein that are linked to an enzyme's catalytic proficiency, while indicating that extensive glycosylation protects the enzyme during pathogenesis by protecting it from protease degradation.

Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser600 inhibits enzyme activity.

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2WT). AtLOX2WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2S600D and Atlox2S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser600 is located at a critical position within the AtLOX2 structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

Fatty acid amide hydrolase and 9-lipoxygenase modulate cotton seedling growth by ethanolamide oxylipin levels.

Polyunsaturated N-acylethanolamines (NAEs) can be hydrolyzed by fatty acid amide hydrolase (FAAH) or oxidized by lipoxygenase (LOX). In Arabidopsis (Arabidopsis thaliana), the 9-LOX product of linoleoylethanolamide, namely, 9-hydroxy linoleoylethanolamide (9-NAE-HOD), is reported to negatively regulate seedling development during secondary dormancy. In upland cotton (Gossypium hirsutum L.), six putative FAAH genes (from two diverged groups) and six potential 9-LOX genes are present; however, their involvement in 9-NAE-HOD metabolism and its regulation of seedling development remain unexplored. Here, we report that in cotton plants, two specific FAAH isoforms (GhFAAH Ib and GhFAAH IIb) are needed for hydrolysis of certain endogenous NAEs. Virus-induced gene silencing (VIGS) of either or both FAAHs led to reduced seedling growth and this coincided with reduced amidohydrolase activities and elevated quantities of endogenous 9-NAE-HOD. Transcripts of GhLOX21 were consistently elevated in FAAH-silenced tissues, and co-silencing of GhLOX21 and GhFAAH (Ib and/or IIb) led to reversal of seedling growth to normal levels (comparable with no silencing). This was concomitant with reductions in the levels of 9-NAE-HOD, but not of 13-NAE-HOD. Pharmacological experiments corroborated the genetic and biochemical evidence, demonstrating that direct application of 9-NAE-HOD, but not 13-NAE-HOD or their corresponding free fatty acid oxylipins, inhibited the growth of cotton seedlings. Additionally, VIGS of GhLOX21 in cotton lines overexpressing AtFAAH exhibited enhanced growth and no detectable 9-NAE-HOD. Altogether, we conclude that the growth of cotton seedlings involves fine-tuning of 9-NAE-HOD levels via FAAH-mediated hydrolysis and LOX-mediated production, expanding the mechanistic understanding of plant growth modulation by NAE oxylipins to a perennial crop species.

Thirty years with three-dimensional structures of lipoxygenases.

The X-ray crystal structures of soybean lipoxygenase (LOX) and rabbit 15-LOX were reported in the 1990s. Subsequent 3D structures demonstrated a conserved U-like shape of the substrate cavities as reviewed here. The 8-LOX:arachidonic acid (AA) complex showed AA bound to the substrate cavity carboxylate-out with C10 at 3.4 Å from the iron metal center. A recent cryo-electron microscopy (EM) analysis of the 12-LOX:AA complex illustrated AA in the same position as in the 8-LOX:AA complex. The 15- and 12-LOX complexes with isoenzyme-specific inhibitors/substrate mimics confirmed the U-fold. 5-LOX oxidizes AA to leukotriene A4, the first step in biosynthesis of mediators of asthma. The X-ray structure showed that the entrance to the substrate cavity was closed to AA by Phe and Tyr residues of a partly unfolded α2-helix. Recent X-ray analysis revealed that soaking with inhibitors shifted the short α2-helix to a long and continuous, which opened the substrate cavity. The α2-helix also adopted two conformations in 15-LOX. 12-LOX dimers consisted of one closed and one open subunit with an elongated α2-helix. 13C-ENDOR-MD computations of the 9-MnLOX:linoleate complex showed carboxylate-out position with C11 placed 3.4 ± 0.1 Å from the catalytic water. 3D structures have provided a solid ground for future research.

Changes in cardiovascular arachidonic acid metabolism in experimental models of menopause and implications on postmenopausal cardiac hypertrophy.

Menopause is a normal stage in the human female aging process characterized by the cessation of menstruation and the ovarian production of estrogen and progesterone hormones. Menopause is associated with an increased risk of several different diseases. Cardiovascular diseases are generally less common in females than in age-matched males. However, this female advantage is lost after menopause. Cardiac hypertrophy is a disease characterized by increased cardiac size that develops as a response to chronic overload or stress. Similar to other cardiovascular diseases, the risk of cardiac hypertrophy significantly increases after menopause. However, the exact underlying mechanisms are not yet fully elucidated. Several studies have shown that surgical or chemical induction of menopause in experimental animals is associated with cardiac hypertrophy, or aggravates cardiac hypertrophy induced by other stressors. Arachidonic acid (AA) released from the myocardial phospholipids is metabolized by cardiac cytochrome P450 (CYP), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes to produce several eicosanoids. AA-metabolizing enzymes and their respective metabolites play an important role in the pathogenesis of cardiac hypertrophy. Menopause is associated with changes in the cardiovascular levels of CYP, COX, and LOX enzymes and the levels of their metabolites. It is possible that these changes might play a role in the increased risk of cardiac hypertrophy after menopause.

A non-invasive method for phenotyping scab-tolerant apple plants using volatile organic compounds.

One of the most devastating diseases of apples is scab, caused by the fungus Venturia inaequalis. Most commercial apple varieties are susceptible to this disease; only a few are resistant. Breeding approaches are being used to develop better apple varieties that are resistant to scab. Volatile organic compounds (VOCs) contribute greatly to a plant's phenotype, and their emission profile largely depends on the genotype. In the non-destructive phenotyping of plants, VOCs can be used as biomarkers. In this study, we assessed non-destructively the scab tolerance potential of resistant (cv. 'Prima') and susceptible (cv. 'Oregon Spur') apple cultivars by comparing their major leaf VOC compositions and relative proportions. A comparison of the leaf VOC profiles of the two cultivars revealed 16 different VOCs, with cis-3-hexenyl acetate (3HA) emerging as a biomarker of cultivar differences. V. inaequalis growth was significantly inhibited in vitro by 3HA treatment. 3HA was significantly effective in reducing scab symptoms on V. inaequalis-inoculated leaves of 'Oregon Spur.' The resistant cultivar 'Prima' also exhibited higher lipoxygenase (LOX) activity and α-linolenic acid (ALA) levels, suggesting that V. inaequalis resistance is linked to LOX activity and 3HA biosynthesis. This study proposes 3HA as a potential biomarker for rapid non-destructive screening of scab-resistant apple germplasm of 'Prima' based on leaf VOCs.

Arabidopsis Transcriptomics Reveals the Role of Lipoxygenase2 (AtLOX2) in Wound-Induced Responses.

In wounded Arabidopsis thaliana leaves, four 13S-lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects.

Synthesis and Antioxidant Activity of N-Benzyl-2-[4-(aryl)-1H-1,2,3-triazol-1-yl]ethan-1-imine Oxides.

The synthesis, antioxidant capacity, and anti-inflammatory activity of four novel N-benzyl-2-[4-(aryl)-1H-1,2,3-triazol-1-yl]ethan-1-imine oxides 10a-d are reported herein. The nitrones 10a-d were tested for their antioxidant properties and their ability to inhibit soybean lipoxygenase (LOX). Four diverse antioxidant tests were used for in vitro antioxidant assays, namely, interaction with the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl radical) as well as with the water-soluble azo compound AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride), competition with DMSO for hydroxyl radicals, and the scavenging of cationic radical ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation). Nitrones 10b, 10c, and 10d, having the 4-fluorophenyl, 2,4-difluorophenyl, and 4-fluoro-3-methylphenyl motif, respectively, exhibited high interaction with DPPH (64.5-81% after 20 min; 79-96% after 60 min), whereas nitrone 10a with unfunctionalized phenyl group showed the lowest inhibitory potency (57% after 20 min, 78% after 60 min). Nitrones 10a and 10d, decorated with phenyl and 4-fluoro-3-methylphenyl motif, respectively, appeared the most potent inhibitors of lipid peroxidation. The results obtained from radical cation ABTS•+ were not significant, since all tested compounds 10a-d showed negligible activity (8-46%), much lower than Trolox (91%). Nitrone 10c, bearing the 2,4-difluorophenyl motif, was found to be the most potent LOX inhibitor (IC50 = 10 µM).

Synthesis and Antioxidant Activities of Novel Pyrimidine Acrylamides as Inhibitors of Lipoxygenase: Molecular Modeling and In Silico Physicochemical Studies.

The pyrimidine ring is present in various biomolecules such as DNA and RNA bases, aminoacids, vitamins, etc. Additionally, many clinically used drugs including methotrexate and risperidone contain the pyrimidine heterocyclic scaffold as well. Pyrimidine derivatives present diverse biological activities including antioxidant and anticancer activities and can be considered as privileged scaffolds in drug discovery for the treatment of various diseases. Piperidine pyrimidine amides have gained significant attention due to their enzymatic inhibitory activity. Based on our experience and ongoing investigation on cinnamic acid derivatives, their hybrids and substituted pteridines acting as lipoxygenase inhibitors, antioxidants, anti-cancer, and anti-inflammatory agents a series of novel piperidine pyrimidine cinnamic acids amides have been designed and synthesized. The novel hybrids were studied for their antioxidant and anti-inflammatory potential. They exhibit moderate antioxidant activity in the DPPH assay which may be related to their bulkiness. Moreover, moderate to good lipid peroxidation inhibition potential was measured. With regards to their lipoxygenase inhibitory activity, however, two highly potent inhibitors out of the nine tested derivatives were identified, demonstrating IC50 values of 10.7 µM and 1.1 µM, respectively. Molecular docking studies to the target enzyme lipoxygenase support the experimental results.

The Composition of Volatiles and the Role of Non-Traditional LOX on Target Metabolites in Virgin Olive Oil from Autochthonous Dalmatian Cultivars.

The lipoxygenase pathway has a significant influence on the composition of the volatile components of virgin olive oil (VOO). In this work, the influence of the maturity index (MI) on the activity of the lipoxygenase enzyme (LOX) in the fruits of the autochthonous Dalmatian olive cultivars Oblica, Levantinka and Lastovka was studied. The analysis of the primary oxidation products of linoleic acid in the studied cultivars showed that LOX synthesises a mixture of 9- and 13-hydroperoxides of octadecenoic acid in a ratio of about 1:2, which makes it a non-traditional plant LOX. By processing the fruits of MI~3, we obtained VOOs with the highest concentration of desirable C6 volatile compounds among the cultivars studied. We confirmed a positive correlation between MI, the enzyme activity LOX and the concentration of hexyl acetate and hexanol in cultivars Oblica and Lastovka, while no positive correlation with hexanol was observed in the cultivar Levantinka. A significant negative correlation was found between total phenolic compounds in VOO and LOX enzyme activity, followed by an increase in the MI of fruits. This article contributes to the selection of the optimal harvest time for the production of VOOs with the desired aromatic properties and to the knowledge of the varietal characteristics of VOOs.

UVR8-TCP4-LOX2 module regulates UV-B tolerance in Arabidopsis.

The phytohormone jasmonate (JA) coordinates stress and growth responses to increase plant survival in unfavorable environments. Although JA can enhance plant UV-B stress tolerance, the mechanisms underlying the interaction of UV-B and JA in this response remain unknown. In this study, we demonstrate that the UV RESISTANCE LOCUS 8 - TEOSINTE BRANCHED1, Cycloidea and PCF 4 - LIPOXYGENASE2 (UVR8-TCP4-LOX2) module regulates UV-B tolerance dependent on JA signaling pathway in Arabidopsis thaliana. We show that the nucleus-localized UVR8 physically interacts with TCP4 to increase the DNA-binding activity of TCP4 and upregulate the JA biosynthesis gene LOX2. Furthermore, UVR8 activates the expression of LOX2 in a TCP4-dependent manner. Our genetic analysis also provides evidence that TCP4 acts downstream of UVR8 and upstream of LOX2 to mediate plant responses to UV-B stress. Our results illustrate that the UV-B-dependent interaction of UVR8 and TCP4 serves as an important UVR8-TCP4-LOX2 module, which integrates UV-B radiation and JA signaling and represents a new UVR8 signaling mechanism in plants.

Anti-inflammatory potential of aconitine produced by endophytic fungus Acremonium alternatum.

Argemone mexicana belonging to family Papaveraceae is a traditional medicinal plant widely utilized by tribal people in India for treating various ailments like skin infections, wounds and inflammation. This plant is very rich in alkaloidal content, which has a great potential in the treatment of anti-inflammatory disorders. Therapeutically promising bioactive molecules are often produced by endophytic fungi associated with medicinal plants. In this investigation, endophytic fungi were isolated from various parts of A. mexicana and screened for alkaloidal content. Among these, one of the fungal isolate, Acremonium alternatum AMEF-5 producing maximum alkaloids showed significant anti-inflammatory activity. Fractionation of this crude fungal extract through column chromatography yielded eight fractions, which were further screened for anti-inflammatory activities. Fraction 3 exhibited significant anti-inflammatory activity by the inhibition of lipoxygenase enzyme (IC50 15.2 ± 0.09 µg/ml), scavenging of the nitric oxide radicals (IC50 11.38 ± 0.35 µg/ml), protein denaturation (IC50 14.93 ± 0.4 µg/ml), trypsin inhibition (IC50 12.06 ± 0.64 µg/ml) and HRBC stabilization (IC50 11.9 ± 0.22 µg/ml). The bioactive alkaloid in fraction 3 was identified as aconitine which was confirmed by UV, FTIR, HPLC, HRMS, 1H NMR, and 13C NMR analysis. This study demonstrates that endophytic fungi serve a potential source for sustainable production of therapeutically important alkaloids.

Biotransformation of Polyunsaturated Fatty Acids to Trioxilins by Lipoxygenase from Pleurotus sajor-caju.

A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629 µmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146 kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB3 , 13,14,15-TrXB4 , and 15,16,17-TrXB5 , respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pH 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10 mM of AA, EPA, and DHA to 8.7 mM of 13,14,15-TrXB3 (conversion rate: 87 %), 7.9 mM of 13,14,15-TrXB4 (79 %), and 7.2 mM of 15,16,17-TrXB5 (72 %) in 15, 20, and 20 min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.

Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4.

[Figure: see text].

Synthesis of 12-aminododecenoic acid by coupling transaminase to oxylipin pathway enzymes.

Biobased polymers derived from plant oils are sustainable alternatives to petro based polymers. In recent years, multienzyme cascades have been developed for the synthesis of biobased ω-aminocarboxylic acids, which serve as building blocks for polyamides. In this work, we have developed a novel enzyme cascade for the synthesis of 12-aminododeceneoic acid, a precursor for nylon-12, starting from linoleic acid. Seven bacterial ω-transaminases (ω-TAs) were cloned, expressed in Escherichia coli and successfully purified by affinity chromatography. Activity towards the oxylipin pathway intermediates hexanal and 12-oxododecenoic acid in their 9(Z) and 10(E) isoforms was demonstrated for all seven transaminases in a coupled photometric enzyme assay. The highest specific activities were obtained with ω-TA from Aquitalea denitrificans (TRAD), with 0.62 U mg-1 for 12-oxo-9(Z)-dodecenoic acid, 0.52 U mg-1 for 12-oxo-10(E)-dodecenoic acid and 1.17 U mg-1 for hexanal. A one-pot enzyme cascade was established with TRAD and papaya hydroperoxide lyase (HPLCP-N), reaching conversions of 59% according to LC-ELSD quantification. Starting from linoleic acid, up to 12% conversion to 12-aminododecenoic acid was achieved with a 3-enzyme cascade comprising soybean lipoxygenase (LOX-1), HPLCP-N and TRAD. Higher product concentrations were achieved by the consecutive addition of enzymes compared to simultaneous addition at the beginning. KEY POINTS: • Seven ω-transaminases converted 12-oxododecenoic acid into its corresponding amine. • A three-enzyme cascade with lipoxygenase, hydroperoxide lyase, and ω-transaminase was established for the first time. • A one-pot transformation of linoleic acid to 12-aminododecenoic acid, a precursor of nylon-12 was achieved.

Isomerization of Oxidized Linoleate Metabolites with trans/cis Conjugated Diene Moiety to Those with trans/trans One in the Lipoxygenase/Linoleate/Hydrogen Polysulfide System.

Endogenous hydrogen polysulfides are radical scavengers, and the resulting thiyl radical may catalyze isomerization of the cis-double bond to a trans-double bond. This study examined whether oxidized linoleate species with trans/trans-conjugated diene moieties were generated in the 15-lipoxygenase/linoleate/hydrogen polysulfide system at a lower oxygen content. When 40 µL of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleate, 1.0 µM soybean 15-lipoxygenase, and 100 µM sodium trisulfide was placed in a 0.6 mL polypropylene microtube for 1 h at 25 °C, the proportion of (E/E)-oxo-octadecadienoic acids (OxoODEs) content to the total OxoODEs content was estimated to be more than 80% (mol/mol). OxoODEs are generated through the pseudoperoxidase reaction of ferrous 15-lipoxygenase with hydroperoxy octadecadienoic acids (HpODEs), which are produced by the lipoxygenase reaction of ferric 15-lipoxygenase. The content of OxoODEs was positively correlated with the content of 9-HpODEs, indicating that 9-HpODEs production is involved in converting ferric 15-lipoxygenase to ferrous 15-lipoxygenase. Furthermore, when 40 µL of 0.1 M phosphate buffer (pH 7.4) containing 1.0 mM linoleate, 1.0 µM soybean 15-lipoxygenase, 100 µM sodium trisulfide, and nitroxyl radical (carbon-centered radical-trapping agent, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-N-oxyl (CmΔP)) was incubated in a 0.6 mL polypropylene microtube at room temperature, CmΔP-(E/Z)-ODEs were isomerized to CmΔP-(E/E)-ODEs in a time-dependent manner and this isomerization was inhibited by a radical scavenger, Trolox. The results indicate that thiyl radicals derived from hydrogen polysulfides isomerize trans/cis conjugated diene moiety to the trans/trans moiety.

Hydroperoxidation of Docosahexaenoic Acid by Human ALOX12 and pigALOX15-mini-LOX.

Human lipoxygenase 12 (hALOX12) catalyzes the conversion of docosahexaenoic acid (DHA) into mainly 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-H(p)DHA). This hydroperoxidation reaction is followed by an epoxidation and hydrolysis process that finally leads to maresin 1 (MaR1), a potent bioactive specialized pro-resolving mediator (SPM) in chronic inflammation resolution. By combining docking, molecular dynamics simulations, and quantum mechanics/molecular mechanics calculations, we have computed the potential energy profile of DHA hydroperoxidation in the active site of hALOX12. Our results describe the structural evolution of the molecular system at each step of this catalytic reaction pathway. Noteworthy, the required stereospecificity of the reaction leading to MaR1 is explained by the configurations adopted by DHA bound to hALOX12, along with the stereochemistry of the pentadienyl radical formed after the first step of the mechanism. In pig lipoxygenase 15 (pigALOX15-mini-LOX), our calculations suggest that 14S-H(p)DHA can be formed, but with a stereochemistry that is inadequate for MaR1 biosynthesis.

Development of Novel Pyrrole Derivatives and Their Cinnamic Hybrids as Dual COX-2/LOX Inhibitors.

Molecular hybridization has emerged as a promising approach in the treatment of diseases exhibiting multifactorial etiology. With regard to this, dual cyclooxygenase-2/lipoxygenase (COX-2/LOX) inhibitors could be considered a safe alternative to traditional non-steroidal anti-inflammatory drugs (tNSAIDs) and selective COX-2 inhibitors (coxibs) for the treatment of inflammatory conditions. Taking this into account, six novel pyrrole derivatives and pyrrole-cinnamate hybrids were developed as potential COX-2 and soybean LOX (sLOX) inhibitors with antioxidant activity. In silico calculations were performed to predict their ADMET (absorption, distribution, metabolism, excretion, toxicity) properties and drug-likeness, while lipophilicity was experimentally determined as RM values. All synthesized compounds (1-4, 5-8) could be described as drug-like. The results from the docking studies on COX-2 were in accordance with the in vitro studies. According to molecular docking studies on soybean LOX, the compounds displayed allosteric interactions with the enzyme. Pyrrole 2 appeared to be the most potent s-LOX inhibitor (IC50 = 7.5 µM). Hybrids 5 and 6 presented a promising combination of in vitro LOX (IC50 for 5 = 30 µM, IC50 for 6 = 27.5 µM) and COX-2 (IC50 for 5 = 0.55 µM, IC50 for 6 = 7.0 µM) inhibitory activities, and therefore could be used as the lead compounds for the synthesis of more effective multi-target agents.

Silencing of a Pseudo-nitzschia arenysensis lipoxygenase transcript leads to reduced oxylipin production and impaired growth.

Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.