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.

[What is the Initiating Reaction for the Lipid Radical Chain Reaction System That Can Induce Ferroptotic Cell Death at the Lower Oxygen Content?]

The neural cell death in cerebral infarction is suggested to be ferroptosis-like cell death, involving the participation of 15-lipoxygenase (15-LOx). Ferroptosis is induced by lipid radical species generated through the one-electron reduction of lipid hydroperoxides, and it has been shown to propagate intracellularly and intercellularly. At lower oxygen concentration, it appeared that both regiospecificity and stereospecificity of conjugated diene moiety in lipoxygenase-catalysed lipid hydroperoxidation are drastically lost. As a result, in the reaction with linoleic acid, the linoleate 9-peroxyl radical-ferrous lipoxygenase complex dissolves into the linoleate 9-peroxyl radical and ferrous 15-lipoxygenase. Subsequently, the ferrous 15-lipoxygenase then undergoes one-electron reduction of 13-hydroperoxy octadecadienoic acid, generating an alkoxyl radical (pseudoperoxidase reaction). A part of the produced lipid alkoxyl radicals undergoes cleavage of C-C bonds, liberating small molecular hydrocarbon radicals. Particularly, in ω-3 polyunsaturated fatty acids, which are abundant in the vascular and nervous systems, the liberation of small molecular hydrocarbon radicals was more pronounced compared to ω-6 polyunsaturated fatty acids. The involvement of these small molecular hydrocarbon radicals in the propagation of membrane lipid damage is suggested.

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.

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.

Comparison between hydrodynamic and ultrasound cavitation on the inactivation of lipoxygenase and physicochemical properties of soy milk.

The effects of hydrodynamic cavitation (HC) and ultrasound cavitation (UC) on the lipoxygenase activity and physicochemical properties of soy milk were evaluated. The results revealed that both ultrasound cavitation and hydrodynamic cavitation significantly inactivated the lipoxygenase activity. After the exposure to ultrasound cavitation at 522.5 W/L and 70 °C for 12 min, the lipoxygenase activity was inactivated by 96.47 %. Meanwhile, HC treatment with the cavitation number of 0.0133 for 240 min led to the loss of 79.31 % of lipoxygenase activity. An artificial neural network was used to model and visualize the effects of different parameters after ultrasound cavitation treatment on the inactivation efficiency of soy milk. Turbiscan test results showed that hydrodynamic and ultrasound cavitation decreased the instability index and particle size of soy milk. Moreover, the total free amino acid content was significantly increased after hydrodynamic and ultrasound cavitation treatment. Gas chromatography-mass spectrometry showed that the total content of beany flavor compounds decreased after acoustic cavitation and HC treatment. Acoustic cavitation and HC affected the tertiary and secondary structure of soy milk, which was related to the inactivation of lipoxygenase. We aim to explore a potential and effective way of the application in soy milk processing by comparing the ultrasound equipped with heat treatment and hydrodymic cavitation.

Identification and characterization of lipoxygenase (LOX) genes involved in abiotic stresses in yellow horn.

Lipoxygenase (LOX) gene plays an essential role in plant growth, development, and stress response. 15 LOX genes were identified, which were unevenly distributed on chromosomes and divided into three subclasses in this study. In promoter region analysis, many cis-elements were identified in growth and development, abiotic stress response, hormonal response, and light response. qRT-PCR showed that the LOX gene showed tissue specificity in seven tissues, especially XsLOX1, 3, and 7 were relatively highly expressed in roots, stems, and axillary buds. The different expression patterns of LOX genes in response to abiotic stress and hormone treatment indicate that different XsLOX genes have different reactions to these stresses and play diversified roles. This study improves our understanding of the mechanism of LOX regulation in plant growth, development, and stress and lays a foundation for further analysis of biological functions.

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.

Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes.

ω-Alkynyl-fatty acids can be used as probes for covalent binding to intracellular macromolecules. To inform future in vivo studies, we determined the rates of reaction of ω-alkynyl-labeled linoleate with recombinant enzymes of the skin 12R-lipoxygenase (12R-LOX) pathway involved in epidermal barrier formation (12R-LOX, epidermal lipoxygenase-3 (eLOX3), and SDR9C7). We also examined the reactivity of ω-alkynyl-arachidonic acid with representative lipoxygenase enzymes employing either "carboxyl end-first" substrate binding (5S-LOX) or "tail-first" (platelet-type 12S-LOX). ω-Alkynyl-linoleic acid was oxygenated by 12R-LOX at 62 ± 9 % of the rate compared to linoleic acid, the alkynyl-9R-HPODE product was isomerized by eLOX3 at only 43 ± 1 % of the natural substrate, whereas its epoxy alcohol product was converted to epoxy ketone linoleic by an NADH-dependent dehydrogenase (SDR9C7) with 91 ± 1 % efficiency. The results suggest the optimal approach will be application of the 12R-LOX/eLOX3-derived epoxyalcohol, which should be most efficiently incorporated into the pathway and allow subsequent analysis of covalent binding to epidermal proteins. Regarding the orientation of substrate binding in LOX catalysis, our results and previous reports suggest the ω-alkynyl group has a stronger inhibitory effect on tail-first binding, as might be expected. Beyond slowing the reaction, however, we found that the tail-first binding and transformation of ω-alkynyl-arachidonic acid by platelet-type 12S-LOX results in almost complete enzyme inactivation, possibly due to reactive intermediates blocking the enzyme active site. Overall, the results reinforce the conclusion that ω-alkynyl-fatty acids are suitable for selected applications after appropriate reactivity is established.

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.

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.

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.

Improving the catalytic efficiency of Pseudomonas aeruginosa lipoxygenase by semi-rational design.

Lipoxygenase (LOX) catalyzes the peroxidation of unsaturated fatty acids to produce hydroperoxides, which had been widely used in food, medicine and chemical industries due to its decoloration of food and conversion of renewable oils. Thus, higher catalytic activity and stability is desired for low-cost and expanded industrial applications of LOX. To improve the catalytic activity of LOX, a mutant library of Pseudomonas aeruginosa lipoxygenase (PaLOX) was firstly built via semi-rational design. The kcat/Km of mutant increased by 9.2-fold and the half-life (t1/2) at 50 °C increased by 4.6 min. Molecular dynamics (MD) simulation indicated that mutation reduced steric hindrance to substrate binding and increased the flexibility of the lid domain that covered the bound unsaturated fatty acid substrate. In addition, van der Waals interactions between the substrate and amino acid residues of the binding pocket increased and alkyl and Pi-alkyl interactions decreased, which might improve the flexibility and substrate binding affinity. These findings promoted understanding of the structure-function relationship of LOX and increase its catalytic efficiency and stability for further industrial application.

Dynamical activation of function in metalloenzymes.

Formulations of hydrogen tunneling in enzyme-catalysed C-H activation reactions indicate enthalpic barriers to reaction that are independent of chemical steps and dependent on the protein scaffold. A tool to identify catalytically relevant site-specific protein thermal networks has emerged from temperature-dependent hydrogen deuterium exchange (TDHDX). Focusing on mutant enzyme forms with altered activation energies for catalysis, TDHDX provides a comparative analysis of the impact of mutation on Ea for local protein unfolding. Identified thermal networks appear unrelated to protein scaffold conservation and track to the dictates of the catalysed reaction, including sites for metal binding. The positions of thermal networks provide a framework for further understanding of time-dependent, functionally relevant protein motions. Measurement of nanosecond Stokes shifts at the surface of the thermal network in soybean lipoxygenase yields activation energies that are identical to Ea values measured for kcat . This finding identifies a rapid (> nanosecond), long-range and cooperative structural reorganization as the thermal barrier to catalysis. A model for protein dynamics is put forward that integrates broadly distributed protein conformational sampling with protein embedded thermal networks.

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.

Lipoxygenase (LOX) in Sweet and Hot Pepper (Capsicum annuum L.) Fruits during Ripening and under an Enriched Nitric Oxide (NO) Gas Atmosphere.

Lipoxygenases (LOXs) catalyze the insertion of molecular oxygen into polyunsaturated fatty acids (PUFA) such as linoleic and linolenic acids, being the first step in the biosynthesis of a large group of biologically active fatty acid (FA)-derived metabolites collectively named oxylipins. LOXs are involved in multiple functions such as the biosynthesis of jasmonic acid (JA) and volatile molecules related to the aroma and flavor production of plant tissues, among others. Using sweet pepper (Capsicum annuum L.) plants as a model, LOX activity was assayed by non-denaturing polyacrylamide gel electrophoresis (PAGE) and specific in-gel activity staining. Thus, we identified a total of seven LOX isozymes (I to VII) distributed among the main plant organs (roots, stems, leaves, and fruits). Furthermore, we studied the FA profile and the LOX isozyme pattern in pepper fruits including a sweet variety (Melchor) and three autochthonous Spanish varieties that have different pungency levels (Piquillo, Padrón, and Alegría riojana). It was observed that the number of LOX isozymes increased as the capsaicin content increased in the fruits. On the other hand, a total of eight CaLOX genes were identified in sweet pepper fruits, and their expression was differentially regulated during ripening and by the treatment with nitric oxide (NO) gas. Finally, a deeper analysis of the LOX IV isoenzyme activity in the presence of nitrosocysteine (CysNO, a NO donor) suggests a regulatory mechanism via S-nitrosation. In summary, our data indicate that the different LOX isozymes are differentially regulated by the capsaicin content, fruit ripening, and NO.

Functional Characterization of Novel Bony Fish Lipoxygenase Isoforms and Their Possible Involvement in Inflammation.

Eicosanoids and related compounds are pleiotropic lipid mediators, which are biosynthesized in mammals via three distinct metabolic pathways (cyclooxygenase pathway, lipoxygenase pathway, epoxygenase pathway). These mediators have been implicated in the pathogenesis of inflammatory diseases and drugs interfering with eicosanoid signaling are currently available as antiphlogistics. Eicosanoid biosynthesis has well been explored in mammals including men, but much less detailed information is currently available on eicosanoid biosynthesis in other vertebrates including bony fish. There are a few reports in the literature describing the expression of arachidonic acid lipoxygenases (ALOX isoforms) in several bony fish species but except for two zebrafish ALOX-isoforms (zfALOX1 and zfALOX2) bony fish eicosanoid biosynthesizing enzymes have not been characterized. To fill this gap and to explore the possible roles of ALOX15 orthologs in bony fish inflammation we cloned and expressed putative ALOX15 orthologs from three different bony fish species (N. furzeri, P. nyererei, S. formosus) as recombinant N-terminal his-tag fusion proteins and characterized the corresponding enzymes with respect to their catalytic properties (temperature-dependence, activation energy, pH-dependence, substrate affinity and substrate specificity with different polyenoic fatty acids). Furthermore, we identified the chemical structure of the dominant oxygenation products formed by the recombinant enzymes from different free fatty acids and from more complex lipid substrates. Taken together, our data indicate that functional ALOX isoforms occur in bony fish but that their catalytic properties are different from those of mammalian enzymes. The possible roles of these ALOX-isoforms in bony fish inflammation are discussed.

Diversified Effects of COVID-19 as a Consequence of the Differential Metabolism of Phospholipids and Lipid Peroxidation Evaluated in the Plasma of Survivors and Deceased Patients upon Admission to the Hospital.

As a result of SARS-CoV-2 infection, inflammation develops, which promotes oxidative stress, leading to modification of phospholipid metabolism. Therefore, the aim of this study is to compare the effects of COVID-19 on the levels of phospholipid and free polyunsaturated fatty acids (PUFAs) and their metabolites produced in response to reactions with reactive oxygen species (ROS) and enzymes (cyclooxygenases-(COXs) and lipoxygenase-(LOX)) in the plasma of patients who either recovered or passed away within a week of hospitalization. In the plasma of COVID-19 patients, especially of the survivors, the actions of ROS and phospholipase A2 (PLA2) cause a decrease in phospholipid fatty acids level and an increase in free fatty acids (especially arachidonic acid) despite increased COXs and LOX activity. This is accompanied by an increased level in lipid peroxidation products (malondialdehyde and 8-isoprostaglandin F2α) and lipid mediators generated by enzymes. There is also an increase in eicosanoids, both pro-inflammatory as follows: thromboxane B2 and prostaglandin E2, and anti-inflammatory as follows: 15-deoxy-Δ-12,14-prostaglandin J2 and 12-hydroxyeicosatetraenoic acid, as well as endocannabinoids (anandamide-(AEA) and 2-arachidonylglycerol-(2-AG)) observed in the plasma of patients who recovered. Moreover, the expression of tumor necrosis factor α and interleukins (IL-6 and IL-10) is increased in patients who recovered. However, in the group of patients who died, elevated levels of N-oleoylethanolamine and N-palmitoylethanolamine are found. Since lipid mediators may have different functions depending on the onset of pathophysiological processes, a stronger pro-inflammatory response in patients who have recovered may be the result of the defensive response to SARS-CoV-2 in survivors associated with specific changes in the phospholipid metabolism, which could also be considered a prognostic factor.

Genome-Wide Association and Expression Analysis of the Lipoxygenase Gene Family in Passiflora edulis Revealing PeLOX4 Might Be Involved in Fruit Ripeness and Ester Formation.

Aroma is an important factor in fruit quality. Passiflora edulis (passion fruit) is popular among consumers because of its rich flavor and nutritional value. Esters are the main components of the volatile aroma of passion fruit. Lipoxygenase (LOX), as the first key enzyme upstream of esters, may play an important role in the formation of passion fruit aroma. In this study, a total of 12 passion fruit LOX (PeLOX) members were screened out based on the Passiflora edulis genome database, which were distributed unevenly on 6 chromosomes, all containing the highly conserved lipoxygenase domain and some containing the PLAT domain. The gene structure, evolutionary analysis and cis-acting elements of the family members were predicted in this study. Transcriptome analysis showed that 12 PeLOX genes had different degrees of response to different abiotic stresses (drought stress, salt stress, cold stress, and high temperature). PeLOX1, PeLOX2, PeLOX7, PeLOX11, and PeLOX12 responded significantly to various abiotic stresses, while PeLOX8 and PeLOX9 had little change in expression in all stresses. Quantitative real-time PCR (qRT-PCR) in six tissues revealed that the 12 PeLOX genes exhibited tissue expression specificity, and the relative expression of most genes were particularly high in the roots, stems, and fruits. Focusing on passion fruit ripening and ester synthesis, the transcriptomic analysis showed that with the increase in fruit development and fruit maturity, the expression levels of PeLOX1, PeLOX9, PeLOX11, and PeLOX12 showed downregulated expression, while PeLOX2 and PeLOX4 showed upregulated expression. In particular, the upregulation trend of PeLOX4 was the most obvious, and the qRT-PCR results were consistent with the transcriptome result. Pearson correlation analysis showed that with the development and ripening of fruit, the expression level of PeLOX4, LOX enzyme activity and total ester content all showed an increasing trend, in particular during the period when the peel was red and shrank (from T2 to T3 stage), the esters' contents increased by 37.4 times; the highest expression levels were all in the T3 period. The results indicated that PeLOX4 may be a candidate gene involved in fruit ripeness and the formation of volatile aroma compounds, with the increase in fruit ripening, the expression level of PeLOX4 increased and the LOX enzyme activity increased accordingly, thereby promoting the synthesis of volatile esters in fruit pulp. Our discovery lays the foundation for the functional study of LOX in passion fruit.

Functional Study of Lipoxygenase-Mediated Resistance against Fusarium verticillioides and Aspergillus flavus Infection in Maize.

Mycotoxin contamination of maize kernels by fungal pathogens like Fusarium verticillioides and Aspergillus flavus is a chronic global challenge impacting food and feed security, health, and trade. Maize lipoxygenase genes (ZmLOXs) synthetize oxylipins that play defense roles and govern host-fungal interactions. The current study investigated the involvement of ZmLOXs in maize resistance against these two fungi. A considerable intraspecific genetic and transcript variability of the ZmLOX family was highlighted by in silico analysis comparing publicly available maize pan-genomes and pan-transcriptomes, respectively. Then, phenotyping and expression analysis of ZmLOX genes along with key genes involved in oxylipin biosynthesis were carried out in a maize mutant carrying a Mu transposon insertion in the ZmLOX4 gene (named UFMulox4) together with Tzi18, Mo17, and W22 inbred lines at 3- and 7-days post-inoculation with F. verticillioides and A. flavus. Tzi18 showed the highest resistance to the pathogens coupled with the lowest mycotoxin accumulation, while UFMulox4 was highly susceptible to both pathogens with the most elevated mycotoxin content. F. verticillioides inoculation determined a stronger induction of ZmLOXs and maize allene oxide synthase genes as compared to A. flavus. Additionally, oxylipin analysis revealed prevalent linoleic (18:2) peroxidation by 9-LOXs, the accumulation of 10-oxo-11-phytoenoic acid (10-OPEA), and triglyceride peroxidation only in F. verticillioides inoculated kernels of resistant genotypes.