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.

De novo transcriptome assembly of a lipoxygenase knock-down strain in the diatom Pseudo-nitzschia arenysensis.

Diatoms are microalgae that live in marine and freshwater environments and are responsible for about 20% of the world's carbon fixation. Population dynamics of these cells is finely regulated by intricate signal transduction systems, in which oxylipins are thought to play a relevant role. These are oxygenated fatty acids whose biosynthesis is initiated by a lipoxygenase enzyme (LOX) and are widely distributed in all phyla, including diatoms. Here, we present a de novo transcriptome obtained from the RNA-seq performed in the diatom species Pseudo-nitzschia arenysensis, using both a wild-type and a LOX-silenced strain, which will represent a reliable reference for comparative analyses within the Pseudo-nitzschia genus and at a broader taxonomic scale. Moreover, the RNA-seq data can be interrogated to go deeper into the oxylipins metabolic pathways.

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.

Duplicated Copy Number Variant of the Maize 9-Lipoxygenase ZmLOX5 Improves 9,10-KODA-Mediated Resistance to Fall Armyworms.

Extensive genome structure variations, such as copy number variations (CNVs) and presence/absence variations, are the basis for the remarkable genetic diversity of maize; however, the effect of CNVs on maize herbivory defense remains largely underexplored. Here, we report that the naturally occurring duplication of the maize 9-lipoxygenase gene ZmLOX5 leads to increased resistance of maize to herbivory by fall armyworms (FAWs). Previously, we showed that ZmLOX5-derived oxylipins are required for defense against chewing insect herbivores and identified several inbred lines, including Yu796, that contained duplicated CNVs of ZmLOX5, referred to as Yu796-2×LOX5. To test whether introgression of the Yu796-2×LOX5 locus into a herbivore-susceptible B73 background that contains a single ZmLOX5 gene is a feasible approach to increase resistance, we generated a series of near-isogenic lines that contained either two, one, or zero copies of the Yu796-2×LOX5 locus in the B73 background via six backcrosses (BC6). Droplet digital PCR (ddPCR) confirmed the successful introgression of the Yu796-2×LOX5 locus in B73. The resulting B73-2×LOX5 inbred line displayed increased resistance against FAW, associated with increased expression of ZmLOX5, increased wound-induced production of its primary oxylipin product, the α-ketol, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA), and the downstream defense hormones regulated by this molecule, 12-oxo-phytodienoic acid (12-OPDA) and abscisic acid (ABA). Surprisingly, wound-induced JA-Ile production was not increased in B73-2×LOX5, resulting from the increased JA catabolism. Furthermore, B73-2×LOX5 displayed reduced water loss in response to drought stress, likely due to increased ABA and 12-OPDA content. Taken together, this study revealed that the duplicated CNV of ZmLOX5 quantitively contributes to maize antiherbivore defense and presents proof-of-concept evidence that the introgression of naturally occurring duplicated CNVs of a defensive gene into productive but susceptible crop varieties is a feasible breeding approach for enhancing plant resistance to herbivory and tolerance to abiotic stress.

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.

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.

Polymorphisms in Cyclooxygenase, Lipoxygenase, and TP53 Genes Predict Colorectal Polyp Risk Reduction by Aspirin in the seAFOod Polyp Prevention Trial.

Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin E2. We investigated whether 35 SNPs in oxylipin metabolism genes such as cyclooxygenase (PTGS) and lipoxygenase (ALOX), as well as 7 SNPs already associated with colorectal cancer risk reduction by aspirin (e.g., TP53; rs104522), modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomized 2 × 2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. 542 (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with nonaspirin users was restricted to rs4837960 (PTGS1) common homozygotes [IRR, 0.69; 95% confidence interval (CI), 0.53-0.90); q = 0.06], rs2745557 (PTGS2) compound heterozygote-rare homozygotes [IRR, 0.60 (0.41-0.88); q = 0.06], rs7090328 (ALOX5) rare homozygotes [IRR 0.27 (0.11-0.64); q = 0.05], rs2073438 (ALOX12) common homozygotes [IRR, 0.57 (0.41-0.80); q = 0.05], and rs104522 (TP53) rare homozygotes [IRR, 0.37 (0.17-0.79); q = 0.06]. No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalized, predictive models of colorectal cancer chemoprevention by aspirin. PREVENTION RELEVANCE: Single-nucleotide polymorphisms in genes controlling lipid mediator signaling may modify the colorectal polyp prevention activity of aspirin. Further investigation is required to determine whether testing for genetic variants can be used to target cancer chemoprevention by aspirin to those who will benefit most.

Lipoxygenase in a Giant Sulfur Bacterium: An Evolutionary Solution for Size and Complexity?

Discovery of Thiomargarita magnifica - an exceptionally large giant sulfur bacterium - urges us to pay additional attention to the giant sulfur bacteria and to revisit our recent bioinformatic finding of lipoxygenases in the representatives of the genus Beggiatoa. These close relatives of Thiomargarita magnifica meet the similar size requirements by forming multicellular structures. We hypothesize that their lipoxygenases are a part of the oxylipin signaling system that provides high level of cell-to-cell signaling complexity which, in turn, enables them to reach large sizes.

Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Computational Insights and In Silico Characterization of a Novel Mini-Lipoxygenase from Nostoc Sphaeroides and Its Application in the Quality Improvement of Steamed Bread.

Lipoxygenase (EC1.13.11.12, LOX) has been potentially used in the food industry for food quality improvement. However, the low activity, poor thermal stability, narrow range of pH stability, as well as undesirable isoenzymes and off-flavors, have hampered the application of current commercial LOX. In this study, a putative mini-lipoxygenase gene from cyanobacteria, Nostoc sphaeroides (NsLOX), was cloned and expressed in E. coli BL21. NsLOX displayed only 26.62% structural identity with the reported LOX from Cyanothece sp., indicating it as a novel LOX. The purified NsLOX showed the maximum activity at pH 8.0 and 15 °C, with superior stability at a pH range from 6.0 to 13.0, retaining about 40% activity at 40 °C for 90 min. Notably, NsLOX exhibited the highest specific activity of 78,080 U/mg towards linoleic acid (LA), and the kinetic parameters-Km, kcat, and kcat/Km-attain values of 19.46 µM, 9199.75 s-1, and 473.85 µM-1 s-1, respectively. Moreover, the activity of NsLOX was obviously activated by Ca2+, but it was completely inhibited by Zn2+ and Cu2+. Finally, NsLOX was supplied in steamed bread and contributed even better improved bread quality than the commercial LOX. These results suggest NsLOX as a promising substitute of current commercial LOX for application in the food industry.

Genome-wide identification and expression pattern analysis of lipoxygenase genes of chickpea (Cicer arietinum L.) in response to accelerated aging.

Seed aging is a major problem which is caused by various factors such as unfavorable physiological, biochemical, and metabolic alterations in seed. Lipoxygenase (LOXs), an oxidoreductase enzyme that catalyzes the oxidation of polyunsaturated fatty acids, acts as a negative regulator in seed viability and vigour during storage. In this study, we identified ten putative LOX gene family members in the chickpea genome, designated as "CaLOX" which are mainly located in the cytoplasm and chloroplast. These genes share different physiochemical properties and similarities in their gene structures and conserved functional regions. The promoter region contained the cis-regulatory elements and transcription binding factors, which were mainly linked to biotic and abiotic stress, hormones, and light responsiveness. In this study, chickpea seeds were treated with accelerated aging treatment for 0, 2, and 4 days at 45 °C and 85 % relative humidity. Increased level of reactive oxygen species, malondialdehyde, electrolyte leakage, proline, lipoxygenase (LOX) activity, and decreased catalase activity indicates cellular dysfunction which demonstrates seed deterioration. Quantitative real-time analysis reveals that 6 CaLOX genes were upregulated, and 4 CaLOX genes were downregulated during the seed aging process in chickpea. This comprehensive study will reveal the role of the CaLOX gene in response to aging treatment. The identified gene may be used to develop better-quality seeds in chickpea.

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.

The lipoxygenase OsLOX10 affects seed longevity and resistance to saline-alkaline stress during rice seedlings.

Prolonged storage of rice seeds can lead to a decrease in seed vigor and seedling quality. The Lipoxygenase (LOX) gene family is widely distributed in plants, and LOX activity is closely related to seed viability and stress tolerance. In this study, the lipoxygenase OsLOX10 gene from the 9-lipoxygenase metabolic pathway was cloned from rice, and its roles in determining seed longevity and tolerance to saline-alkaline stress caused by Na2CO3 in rice seedlings were mainly investigated. CRISPR/Cas9 knockout of OsLOX10 increased seed longevity compared with the wild-type and OsLOX10 overexpression lines in response to artificial aging. The expression levels of other 9-lipoxygenase metabolic pathway related genes, such as LOX1, LOX2 and LOX3, were increased in the LOX10 overexpression lines. Quantitative real-time PCR and histochemical staining analysis showed that the expression of LOX10 was highest in seed hulls, anthers and the early germinating seeds. KI-I2 staining of starch showed that LOX10 could catalyze the degradation of linoleic acid. Furthermore, we found that the transgenic lines overexpressing LOX10 showed better tolerance to saline-alkaline stress than the wild-type and knockout mutant lines. Overall, our study demonstrated that the knockout LOX10 mutant increased seed longevity, whereas overexpression of LOX10 enhanced tolerance to saline-alkaline stress in rice seedlings.

Genome-wide identification and characterization of lipoxygenase genes related to the English grain aphid infestation response in wheat.

MAIN CONCLUSION: 44 wheat LOX genes were identified by silico genome-wide search method. TaLOX5, 7, 10, 24, 29, 33 were specifically expressed post aphid infestation, indicating their participation in wheat-aphid interaction. In plants, LOX genes play important roles in various biological progresses including seed germination, tuber development, plant vegetative growth and most crucially in plant signal transduction, stress response and plant defense against plant diseases and insects. Although LOX genes have been characterized in many species, the importance of the LOX family in wheat has still not been well understood, hampering further improvement of wheat under stress conditions. Here, we identified 44 LOX genes (TaLOXs) in the whole wheat genome and classified into three subfamilies (9-LOXs, Type I 13-LOXs and Type II 13-LOXs) according to phylogenetic relationships. The TaLOXs belonging to the same subgroup shared similar gene structures and motif organizations. Synteny analysis demonstrated that segmental duplication events mainly contributed to the expansion of the LOX gene family in wheat. The results of protein-protein interaction network (PPI) and miRNA-TaLOXs predictions revealed that three TaLOXs (TaLOX20, 22 and 37) interacted mostly with proteins related to methyl jasmonate (MeJA) signaling pathway. The expression patterns of TaLOXs in different tissues (root, stem, leaf, spike and grain) under diverse abiotic stresses (heat, cold, drought, drought and heat combined treatment, and salt) as well as under diverse biotic stresses (powdery mildew pathogen, Fusarium graminearum and stripe rust pathogen) were systematically analyzed using RNA-seq data. We obtained aphid-responsive candidate genes by RNA-seq data of wheat after the English grain aphid infestation. Aphid-responsive candidate genes, including TaLOX5, 7, 10, 24, 29 and 33, were up-regulated in the wheat aphid-resistant genotype (Lunxuan144), while they were little expressed in the susceptible genotype (Jimai22) during late response (48 h and 72 h) to the English grain aphid infestation. Meanwhile, qRT-PCR analysis was used to validate these aphid-responsive candidate genes. The genetic divergence and diversity of all the TaLOXs in bread wheat and its relative species were investigated by available resequencing data. Finally, the 3D structure of the TaLOX proteins was predicted based on the homology modeling method. This study not only systematically investigated the characteristics and evolutionary relationships of TaLOXs, but also provided potential candidate genes in response to the English grain aphid infestation and laid the foundation to further study the regulatory roles in the English grain aphid infestation of LOX family in wheat and beyond.

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.

Effect of atorvastatin on lipoxygenase pathway-related gene expression in an in vitro model of lipid accumulation in hepatocytes.

Lipid accumulation in hepatocytes can result from an imbalance between lipid acquisition and lipid catabolism. In recent years, it has been discovered that eicosanoids derived from arachidonic acid (AA) have the potential to create specialized pro-resolving lipid mediators to actively resolve inflammation, but it is not clear whether AA and lipoxygenases exert effects on hepatic inflammation. Here, the effects of atorvastatin on the expression of cytoplasmic phospholipase A2 (cPLA2) and lipoxygenase pathway genes (ALOX5, ALOX12, ALOX15, and ALOX15B) were evaluated in an in vitro model of palmitic acid (PA)-induced hepatocyte lipid accumulation in McA-RH7777 (McA) cells. Palmitic acid increased cPLA2 expression, intracellular AA levels, and ALOX12 expression (P < 0.05). Atorvastatin at various concentrations had no significant effects on AA levels or on cPLA2, ALOX15, and ALOX15B expressions. ALOX5 was not detected, despite multiple measurements. Pro-inflammatory IL-1ß expression levels were upregulated by PA (P < 0.01) and attenuated by atorvastatin (P < 0.001). TNFα did not differ among groups. The expression levels of anti-inflammatory IL-10 decreased in response to PA (P < 0.05), but were not affected by atorvastatin. In conclusion, in an in vitro model of lipid accumulation in McA cells, atorvastatin reduced IL-1ß; however, its effect was not mediated by AA and the lipoxygenase pathway at the established doses and treatment duration. Further research is required to investigate time-response data, as well as other drugs and integrated cell systems that could influence the lipoxygenase pathway and modulate inflammation in liver diseases.

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.

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.

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.