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9-Carbon aldehydes such as (2E)-nonenal, (3Z)-nonenal, and (2E,6Z)-nonadienal are important melon and cucumber fragrance compounds. Currently, these molecules are produced either synthetically, which faces consumer aversion, or through biotransformation using plant-extracted enzymes, which is costly and inefficient. In this study, we constructed a Saccharomyces cerevisiae platform for the whole cell biotransformation of polyunsaturated fatty acids (PUFAs) to 9-carbon aldehydes. Heterologous expression of lipoxygenase (LOX) from Nicotiana benthamiana and hydroperoxide lyase (HPL) from Cucumis melo (melon) in S. cerevisiae enabled the production of (2E)-nonenal from readily available polyunsaturated fatty acid substrates. A 5.5-fold increase in (2E)-nonenal titer was then achieved utilizing genetic and reaction condition enhancement strategies. The highest titer of (2E)-nonenal was more than 0.11 mM, with about 9% yield. This platform can potentially be used to produce a variety of other aldehyde products by customizing with LOX and HPL enzymes of different regio-selectivities. KEY POINTS: • Establishment of a S. cerevisiae whole-cell biotransformation platform for cost-efficient, high-yield conversion of PUFAs into high value 9-carbon aldehyde compounds • 5.5-Fold improvement of (2E)-nonenal titer to > 0.11 mM achieved by enhancing reaction conditions and gene expression levels of LOX and HPL., (© 2024. The Author(s).)

Lipoxygenases (LOXs) from lower organisms have substrate flexibility and function versatility in fatty acid oxidation, but it is not clear how these LOXs acquired the ability to execute multiple functions within only one catalytic domain. This work studied a multifunctional LOX from red alga Pyropia haitanensis (PhLOX) which combined hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activity in its active pocket. Molecular docking and site-directed mutagenesis revealed that Phe642 and Phe826 jointly regulated the double peroxidation of fatty acid, Gln777 and Asn575 were essential to the AOS function, and the HPL activity was improved when Asn575, Gln777, or Phe826 was replaced by leucine. Phylogenetic analysis indicated that Asn575 and Phe826 were unique amino acid sites in the separated clades clustered with PhLOX, whereas Phe642 and Gln777 were conserved in plant or animal LOXs. The N-terminal START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain of PhLOX was another key variable, as the absence of this domain disrupted the versatility of PhLOX. Moreover, the functions of two homologous LOXs from marine bacterium Shewanella violacea and red alga Chondrus crispus were examined. The HPL activity of PhLOX appeared to be inherited from a common ancestor, and the AOS function was likely acquired through mutations in some key residues in the active pocket. Taken together, our results suggested that some LOXs from red algae attained their versatility by amalgamating functional domains of ancestral origin and unique amino acid mutations.

Nine-carbon aldehydes and their relative alcohols (C9 aromas) are the main aroma compounds of cucumber (Cucumis sativus L.) fruits and provide a unique cucumber-like note. However, the key regulators of C9 aroma accumulation in cucumber fruit are poorly characterized. Based on C9 aroma dynamic analysis and transcriptome analysis during fruit development of two different cucumber inbred lines, Q16 and Q24, Lipoxygenase09 (CsLOX09) was identified as a candidate gene for C9 aroma accumulation. Additionally, Q24 with higher CsLOX09 expression accumulated more C9 aromas than Q16. To verify the function of CsLOX09, Cslox09 homozygote knockout lines were created. C9 aroma content decreased by 80.79% to 99.16% in these mutants compared to the wild type. To further explore the reasons for the difference in CsLOX09 expression between Q16 and Q24 fruits, a co-expression network was constructed by integrating the C9 aroma-associated metabolism and transcriptomic data. Eighteen candidate transcription factors were highly correlated with the expression of CsLOX09. DNA binding with One Finger 1.8 (CsDof1.8) was confirmed to bind directly to the A/TAAAG motif of the CsLOX09 promoter through dual-luciferase, yeast one-hybrid, chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. Furthermore, C9 aroma content and CsLOX09 expression were significantly increased in the CsDof1.8 overexpression lines. Overall, these data elucidate the metabolic regulation of C9 aromas in cucumber and provide a foundation for facilitating the regulation of flavor in cucumber breeding., Competing Interests: Conflict of interest statement. The authors declare that they have no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Immune-priming occurs in insects after a prior pathogen exposure. However, its underlying mechanism in insects remains elusive. In the present work, immune-priming was detected in a lepidopteran insect, Spodoptera exigua. Specifically, a prior infection with a heat-killed pathogenic bacterium, Escherichia coli, led to increased survival upon the second infection of different pathogens. Plasma collected from larvae with the prior infection possessed the immune-priming factor(s) that significantly up-regulated cellular and humoral immune responses of naïve larvae. Our study also finds that variations in the timing of plasma collection for priming larvae resulted in distinct impacts on both cellular and humoral responses. However, when the active plasma exhibiting the immune-priming was heat-treated, it lost this priming activity, therefore suggesting that protein factor(s) play a role in this immune-priming. An immunofluorescence assay showed that the hemocytes collected from the immune-primed larvae highly reacted to a polyclonal antibody specific to a vertebrate lipocalin, apolipoprotein D (ApoD). Among 27 ApoD genes (Se-ApoD1 ∼ Se-ApoD27) of S. exigua, Se-ApoD3 was found to be highly induced during the immune-priming, in which it was shown to be expressed in hemocytes and fat body from a fluorescence in situ hybridization analysis. RNA interference of Se-ApoD3 expression significantly impaired the immune-priming of S. exigua larvae. Moreover, the inhibition of eicosanoid biosynthesis suppressed the immune-priming, in which treatment with a lipoxygenase (LOX) inhibitor-and not treatment with a cyclooxygenase inhibitor-suppressed immune-priming. Further, an addition of LOX product such as lipoxin A 4 or lipoxin B 4 significantly rescued the lost immune-priming activity. Taken together, these results suggest that a complex of ApoD3 and LOX product mediates the immune-priming activity of S. exigua., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD + ) is a redox cofactor and signal central to cell metabolisms. Disrupting NAD homeostasis in plant alters growth and stress resistance, yet the underlying mechanisms remain largely unknown. Here, by combining genetics with multi-omics, we discover that NAD + deficiency in qs-2 caused by mutation in NAD + biosynthesis gene-Quinolinate Synthase retards growth but induces biosynthesis of defense compounds, notably aliphatic glucosinolates that confer insect resistance. The elevated defense in qs-2 is resulted from activated jasmonate biosynthesis, critically hydroperoxidation of α-linolenic acid by the 13-lipoxygenase (namely LOX2), which is escalated via the burst of chloroplastic ROS-singlet oxygen ( 1 O 2 ). The NAD + deficiency-mediated JA induction and defense priming sequence in plants is recapitulated upon insect infestation, suggesting such defense mechanism operates in plant stress response. Hence, NAD homeostasis is a pivotal metabolic checkpoint that may be manipulated to navigate plant growth and defense metabolism for stress acclimation., (© 2024. The Author(s).)

In wounded Arabidopsis thaliana leaves, four 13 S -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.

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., (© 2024. The Author(s).)

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 , Mo LOX. 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 Mo LOX 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 Mo LOX 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.

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., (© 2024 Scandinavian Plant Physiology Society.)

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., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

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.

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., Competing Interests: The author(s) declare no conflict of interest.

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., (©2023 The Authors; Published by the American Association for Cancer Research.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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- K m , k cat , and k cat / K m -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 Ca 2+ , but it was completely inhibited by Zn 2+ and Cu 2+ . 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.

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-1β 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-1β; 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., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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 (TR AD ), 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 TR AD and papaya hydroperoxide lyase (HPL CP-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), HPL CP-N and TR AD . 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., (© 2023. The Author(s).)

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.

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 Ser 600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2 WT ). AtLOX2 WT 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 Ser 600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2 S600D and Atlox2 S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2 S600A 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 Ser 600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser 600 is located at a critical position within the AtLOX2 structure and highlights how Ser 600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser 600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Na 2 CO 3 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-I 2 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., (© 2023. The Author(s).)

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 E a 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 E a values measured for k cat . 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., (© 2022 Federation of European Biochemical Societies.)

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.

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.

Analyses of fungal genomes of escalate from biological and evolutionary investigations. The biochemical analyses of putative enzymes will inevitably lag behind and only a selection will be characterized. Plant-pathogenic fungi secrete manganese-lipoxygenases (MnLOX), which oxidize unsaturated fatty acids to hydroperoxides to support infection. Six MnLOX have been characterized so far including the 3D structures of these enzymes of the Rice blast and the Take-all fungi. The goal was to use this information to evaluate MnLOX-related gene transcripts to find informative specimens for further studies. Phylogenetic analysis, determinants of catalytic activities, and the C-terminal amino acid sequences divided 54 transcripts into three major subfamilies. The six MnLOX belonged to the same "prototype" subfamily with conserved residues in catalytic determinants and C-terminal sequences. The second subfamily retained the secretion mechanism, presumably necessary for uptake of Mn 2+ , but differed in catalytic determinants and by cysteine replacement of an invariant Leu residue for positioning ("clamping") of fatty acids. The third subfamily contrasted with alanine in the Gly/Ala switch for regiospecific oxidation and a minority contained unprecedented C-terminal sequences or lacked secretion signals. With these exceptions, biochemical analyses of transcripts of the three subfamilies appear to have reasonable prospects to find active enzymes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

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.

Human 15-lipoxygenases (LOX) are critical enzymes in the inflammatory process, producing various pro-resolution molecules, such as lipoxins and resolvins, but the exact role each of the two 15-LOXs in these biosynthetic pathways remains elusive. Previously, it was observed that h15-LOX-1 reacted with 5S-HETE in a non-canonical manner, producing primarily the 5S,12S-diHETE product. To determine the active site constraints of h15-LOX-1 in achieving this reactivity, amino acids involved in the fatty acid binding were investigated. It was observed that R402L did not have a large effect on 5S-HETE catalysis, but F414 appeared to π-π stack with 5S-HETE, as seen with AA binding, indicating an aromatic interaction between a double bond of 5S-HETE and F414. Decreasing the size of F352 and I417 shifted oxygenation of 5S-HETE to C12, while increasing the size of these residues reversed the positional specificity of 5S-HETE to C15. Mutants at these locations demonstrated a similar effect with 7S-HDHA as the substrate, indicating that the depth of the active site regulates product specificity for both substrates. Together, these data indicate that of the three regions proposed to control positional specificity, π-π stacking and active site cavity depth are the primary determinants of positional specificity with 5S-HETE and h15-LOX-1. Finally, the altered reactivity of h15-LOX-1 was also observed with 5S-HEPE, producing 5S,12S-diHEPE instead of 5S,15S-diHEPE (aka resolvin E 4 (RvE 4 ). However, h15-LOX-2 efficiently produces 5S,15S-diHEPE from 5S-HEPE. This result is important with respect to the biosynthesis of the RvE 4 since it obscures which LOX isozyme is involved in its biosynthesis. Future work detailing the expression levels of the lipoxygenase isoforms in immune cells and selective inhibition during the inflammatory response will be required for a comprehensive understanding of RvE 4 biosynthesis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 UFMu lox4 ) 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 UFMu lox4 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.

Human 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis, mostly expressed in leukocytes and thus a crucial component of the innate immune system. In this study, we show that 5-LO, besides its canonical function as an arachidonic acid metabolizing enzyme, is a regulator of gene expression associated with euchromatin. By Crispr-Cas9-mediated 5-LO knockout (KO) in MonoMac6 (MM6) cells and subsequent RNA-Seq analysis, we identified 5-LO regulated genes which could be clustered to immune/defense response, cell adhesion, transcription and growth/developmental processes. Analysis of differentially expressed genes identified cyclooxygenase-2 (COX2, PTGS2) and kynureninase (KYNU) as strongly regulated 5-LO target genes. 5-LO knockout affected MM6 cell adhesion and tryptophan metabolism via inhibition of the degradation of the immunoregulator kynurenine. By subsequent FAIRE-Seq and 5-LO ChIP-Seq analyses, we found an association of 5-LO with euchromatin, with prominent 5-LO binding to promoter regions in actively transcribed genes. By enrichment analysis of the ChIP-Seq results, we identified potential 5-LO interaction partners. Furthermore, 5-LO ChIP-Seq peaks resemble patterns of H3K27ac histone marks, suggesting that 5-LO recruitment mainly takes place at acetylated histones. In summary, we demonstrate a noncanonical function of 5-LO as transcriptional regulator in monocytic cells., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Turnip ( Brassica rapa L. subsp. rapa ) is an important crop with edible and medicinal values, and various stresses, especially salt stress and drought stress, seriously threaten the yield of turnips. LOXs play important roles in regulating plant growth and development, signal transduction, and biotic and abiotic stress responses through secondary metabolites produced by the oxylipin metabolic pathway, and although the turnip genome has been published, however, the role of LOX family genes in various abiotic stress responses has not been systematically studied in turnips. In this study, a total of 15 LOX genes ( BrrLOX ) were identified in turnip, distributed on six chromosomes. Phylogenetic tree analysis classified these LOX genes into two classes: three 9-LOX proteins and 12 13-LOX type II proteins. Gene duplication analysis showed that tandem and segmental duplication were the main pathways for the expansion of the BrrLOX gene family. The Ka and Ks values of the duplicated genes indicate that the BrrLOX gene underwent strong purifying selection. Further analysis of the cis-acting elements of the promoters suggested that the expression of the BrrLOX gene may be influenced by stress and phytohormones. Transcriptome data analysis showed that 13 BrrLOX genes were expressed at one or more stages of turnip tuber development, suggesting that LOX genes may be involved in the formation of turnip fleshy roots. The qRT-PCR analysis showed that four stresses (salt stress, drought stress, cold stress, and heat stress) and three hormone treatments (methyl jasmonate, salicylic acid, and abscisic acid) affected the expression levels of BrrLOX genes and that different BrrLOX genes responded differently to these stresses. In addition, weighted gene co-expression network analysis (WGCNA) of BrrLOX revealed seven co-expression modules, and the genes in these co-expression modules are collectively involved in plant growth and development and stress response processes. Thus, our results provide valuable information for the functional identification and regulatory mechanisms of BrrLOX in turnip growth and development and stress response., Competing Interests: The authors declare there are no competing interests., (©2022 Yan et al.)

Double-oxygenating lipoxygenase (LOX) converted C20- and C22-polyunsaturated fatty acids (PUFAs) into C20 dihydroxy fatty acids (DiHFAs) as inflammatory mediators and C22 DiHFAs as specialized pro-resolving mediators, which are involved in the resolution of inflammation and infection in humans, and their isomers, respectively. However, the quantitative bioconversion of C20- and C22-PUFAs into 9S,15S- and 11S,17S-DiHFAs has been not attempted to date, respectively. Here, we performed the efficient quantitative production of 9S,15S- and 11S,17S-DiHFAs by Escherichia coli expressing 9S-LOX from Sphingopyxis macrogoltabida. The optimal bioconversion conditions of the C20 PUFA arachidonic acid or the C22-PUFA docosahexaenoic acid were pH 8.5, 35 °C, 6 mM substrate, and 5 g dry cells/L for C20 PUFAs or 7 g dry cells/L for C22-PUFAs, respectively. Under these conditions, E. coli expressing double-oxygenating 9S-LOX from S. macrogoltabida converted arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid n-3 , and docosahexaenoic acid into 5.85 mM 9S,15S-dihydroxyeicosatetraenoic acid, 5.79 mM 9S,15S-dihydroxyeicosapentaenoic acid, 5.89 mM 11S,17S-hydroxydocosapentaenoic acid n-3 , and 5.24 mM 11S,17S-dihydroxydocosahexaenoic acid in 40, 30, 50, and 60 min, with conversion yields of 97.5%, 96.5%, 98.1%, and 87.3%; and volumetric productivities of 8.78, 11.6, 7.07, and 5.24 mM/h, respectively. To date, these are the highest concentrations, conversion yields, and volumetric productivities reported in the bioconversion of C20- and C22-PUFAs into DiHFAs., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low, except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were targeted to plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in crude leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific sub-cellular localization of these two enzymes and incubation of crude leaf extracts, which liberates α-linolenic acid via breakdown of endogenous lipids., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved., Results: Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL -1 in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively., Conclusions: In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production., (© 2022. The Author(s).)

Plant lipoxygenases (LOXs) oxygenate linoleic and linolenic acids, creating hydroperoxy derivatives, and from these, jasmonates and other oxylipins are derived. Despite the importance of oxylipin signaling, its activation mechanism remains largely unknown. Here, we show that soybean ACYL-COA-BINDING PROTEIN3 (ACBP3) and ACBP4, two Class II acyl-CoA-binding proteins, suppressed activity of the vegetative LOX homolog VLXB by sequestering it at the endoplasmic reticulum. The ACBP4-VLXB interaction was facilitated by linoleoyl-CoA and linolenoyl-CoA, which competed with phosphatidic acid (PA) for ACBP4 binding. In salt-stressed roots, alternative splicing produced ACBP variants incapable of VLXB interaction. Overexpression of the variants enhanced LOX activity and salt tolerance in Arabidopsis and soybean hairy roots, whereas overexpressors of the native forms exhibited reciprocal phenotypes. Consistently, the differential alternative splicing pattern in two soybean genotypes coincided with their difference in salt-induced lipid peroxidation. Salt-treated soybean roots were enriched in C32:0-PA species that showed high affinity to Class II ACBPs. We conclude that PA signaling and alternative splicing suppress ligand-dependent interaction of Class II ACBPs with VLXB, thereby triggering lipid peroxidation during salt stress. Hence, our findings unveil a dual mechanism that initiates the onset of oxylipin signaling in the salinity response., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Bactrocera oleae , the olive fruit fly, is one of the most important pests affecting the olive fruit, causing serious quantitative and qualitative damage to olive oil production. In this study, the changes induced by B. oleae infestation in the biosynthesis of volatile and phenolic compounds in olive (cvs. Picual, Manzanilla, and Hojiblanca) have been analyzed. Despite cultivar differences, the oils obtained from infested fruits showed a significant increase in the content of certain volatile compounds such as ( E) -hex-2-enal, ethanol, ethyl acetate, and β-ocimene and a drastic decrease of the phenolic contents. The impact of those changes on the inferred quality of the oils has been studied. In parallel, the changes induced by the attack of the olive fly on the expression of some key genes in the biosynthesis of volatile and phenolic compounds, such as lipoxygenase, β-glucosidase, and polyphenol oxidase, have been analyzed. The strong induction of a new olive polyphenol oxidase gene ( OePPO2) explains the reduction of phenolic content in the oils obtained from infested fruits and suggest the existence of a PPO-mediated oxidative defense system in olives.

Background and Aims: Plant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant-P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria., Methods: Tobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches., Key Results: The obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity., Conclusions: Our results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products)., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The C6 aldehydes, alcohols, and the corresponding esters are the most important compounds of virgin olive oil aroma. These C6 volatile compounds are synthesized via the 13-hydroperoxide lyase (13-HPL) branch of the lipoxygenase pathway. In this investigation, a functional analysis of the olive (Olea europaea L.) 13-HPL gene by its overexpression and silencing in olive transgenic lines was carried out. With this aim, sense and RNAi constructs of the olive 13-HPL gene were generated and used for the transformation of embryogenic olive cultures. Leaves from overexpressing lines showed a slight increase in 13-HPL gene expression, whereas RNAi lines exhibited a strong decrease in their transcript levels. Quantification of 13-HPL activity in two overexpressing and two RNAi lines showed a positive correlation with levels of transcripts. Interestingly, RNAi lines showed a high decrease in the content of C6 volatiles linked to a strong increase of C5 volatile compounds, altering the volatile profile in the leaves. In addition, the silencing of the 13-HPL gene severely affected plant growth and development. This investigation demonstrates the role of the 13-HPL gene in the biogenesis of olive volatile compounds and constitutes a functional genomics study in olive related to virgin olive oil quality., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Key Message: Proteomic and lipidomics analyses of WT and GmDGAT1-2 transgenic soybeans showed that GmDGAT1-2 over-expression induced lipoxygenase down-regulatation and oleoin up-regulatation, which significantly changed the compositions and total fatty acid. The main goal of soybean breeding is to increase the oil content. Diacylglycerol acyltransferase (DGAT) is a key rate-limiting enzyme in fatty acid metabolism and may regulate oil content. Herein, 10 GmDGAT genes were isolated from soybean and transferred into wild-type (WT) Arabidopsis. The total fatty acid was 1.2 times higher in T 3 GmDGAT1-2 transgenic Arabidopsis seeds than in WT. Therefore, GmDGAT1-2 was transferred into WT soybean (JACK), and four T 3 transgenic soybean lines were obtained. The results of high-performance gas chromatography and Soxhlet extractor showed that, compared with those of JACK, oleic acid (18:1), and total fatty acid levels in transgenic soybean plants were much higher, but linoleic acid (18:2) was lower than WT. Palmitic acid (16:0), stearic acid (18:0), and linolenic acid (18:3) were not significantly different. For mechanistic studies, 436 differentially expressed proteins (DEPs) and 180 differentially expressed metabolites (DEMs) were identified between WT (JACK) and transgenic soybean pods using proteomic and lipidomics analyses. Four lipoxygenase proteins were down-regulated in linoleic acid metabolism while four oleosin proteins were up-regulated in the final oil formation. The results showed an increase in the total fatty acid and 18:1 composition, and a decrease in the 18:2 composition of fatty acid. Our study brings new insights into soybean genetic transformation and the deep study of molecular mechanism that changes the total fatty acid, 18:1, and 18:2 compositions in GmDGAT1-2 transgenic soybean., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lipoxygenases (Loxs) are dioxygenases that play an important role in plant growth and defense. Loxs affect flour processing quality in common wheat (Triticum aestivum). We conducted a genome-wide association study (GWAS) that identified 306 significant single-nucleotide polymorphisms (SNPs) related to Lox activity in Chinese wheat accessions. Among them, a novel lipoxygenase-encoding (Lpx) gene, TaLpx-B4, was detected on chromosome 3B in a biparental population. Analysis of mutant wheat lines induced using ethyl methanesulfonate confirmed the role of TaLpx-B4 in modulating Lox activity. A phylogenetic tree of various plant Lpx genes indicated the predominance of the 9-Lpx type in common wheat. Further analysis revealed conserved intron number, exon length, and motif number in the TaLpx gene family. GWAS, linkage mapping, and gene annotation collectively showed that 14 out of 29 annotated TaLpx genes played a critical role in regulating Lox activity in the Chinese wheat accessions. Transgenic wheat grains with knockdown of Lpx family genes by RNAi showed significantly lower Lox activity than the wild type. One TaLpx-RNAi line had significantly reduced starch content and dough stability, and thus possessed relatively superior biscuit quality in soft wheat. Further analysis of the transcriptome, lipid components, and other metabolites revealed that knockdown of TaLpx genes significantly increased biscuit quality via changes in unsaturated fatty acid content as well as in starch, sucrose, and galactose metabolism. Our results provide new insights into the role of the TaLpx gene family that will be beneficial in improving soft wheat flour quality., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Teosinte branched1/cycloidea/proliferating cell factor ( TCP ) gene family members are plant-specific transcription factors that regulate plant growth and development by controlling cell proliferation and differentiation. However, there are no reported studies on the TCP gene family in Dendrobium catenatum Lindl. Here, a genome-wide analysis of TCP genes was performed in D. catenatum , and 25 TCP genes were identified. A phylogenetic analysis classified the family into two clades: Class I and Class II. Genes in the same clade share similar conserved motifs. The GFP signals of the DcaTCP-GFPs were detected in the nuclei of tobacco leaf epidermal cells. The activity of DcaTCP4 , which contains the miR319a-binding sequence, was reduced when combined with miR319a. A transient activity assay revealed antagonistic functions of Class I and Class II of the TCP proteins in controlling leaf development through the jasmonate-signaling pathway. After different phytohormone treatments, the DcaTCP genes showed varied expression patterns. In particular, DcaTCP4 and DcaTCP9 showed opposite trends after 3 h treatment with jasmonate. This comprehensive analysis provides a foundation for further studies on the roles of TCP genes in D. catenatum .

Verticillium wilt is a major limiting factor for sustainable production of cotton but the mechanism of controlling this disease is still poorly understood. Lipoxygenase (LOX)-derived oxylipins have been implicated in defense responses against diverse pathogens; however there is limited information about the functional characterization of LOXs in response to Verticillium dahliae infection. In this study, we report the characterization of a cotton LOX gene, GhLOX2, which phylogenetically clustered into 13-LOX subfamily and is closely related to Arabidopsis LOX2 gene. GhLOX2 was predominantly expressed in leaves and strongly induced following V. dahliae inoculation and treatment of methyl jasmonate (MeJA). RNAi-mediated knock-down of GhLOX2 enhanced cotton susceptibility to V. dahliae and was coupled with suppression of jasmonic acid (JA)-related genes both after inoculation with the cotton defoliating strain V991 or MeJA treatment. Interestingly, lignin contents, transcripts of lignin synthesis genes and H 2 O 2 contents were also decreased in GhLOX2-silenced plants. This study suggests that GhLOX2 is involved in defense responses against infection of V. dahliae in cotton and supports that JA is one of the major defense hormones against this pathogen., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The LOX genes have been identified and characterized in many plant species, but studies on the banana LOX genes are very limited. In this study, we respectively identified 18 MaLOX, 11 MbLOX, and 12 MiLOX genes from the Musa acuminata, M. balbisiana and M. itinerans genome data, investigated their gene structures and characterized the physicochemical properties of their encoded proteins. Banana LOXs showed a preference for using and ending with G/C and their encoded proteins can be classified into 9-LOX, Type I 13-LOX and Type II 13-LOX subfamilies. The expansion of the MaLOXs might result from the combined actions of genome-wide, tandem, and segmental duplications. However, tandem and segmental duplications contribute to the expansion of MbLOXs. Transcriptome data based gene expression analysis showed that MaLOX1, 4, and 7 were highly expressed in fruit and their expression levels were significantly regulated by ethylene. And 11, 12 and 7 MaLOXs were found to be low temperature-, high temperature-, and Fusarium oxysporum f. sp. Cubense tropical race 4 (FocTR4)-responsive, respectively. MaLOX8, 9 and 13 are responsive to all the three stresses, MaLOX4 and MaLOX12 are high temperature- and FocTR4-responsive; MaLOX6 and MaLOX17 are significantly induced by low temperature and FocTR4; and the expression of MaLOX7 and MaLOX16 are only affected by high temperature. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression levels of several MaLOXs are regulated by MeJA and FocTR4, indicating that they can increase the resistance of banana by regulating the JA pathway. Additionally, the weighted gene co-expression network analysis (WGCNA) of MaLOXs revealed 3 models respectively for 5 (MaLOX7-11), 3 (MaLOX6, 13, and 17), and 1 (MaLOX12) MaLOX genes. Our findings can provide valuable information for the characterization, evolution, diversity and functionality of MaLOX, MbLOX and MiLOX genes and are helpful for understanding the roles of LOXs in banana growth and development and adaptations to different stresses.

Aberrant expression or dysfunction of a number of genes in the brain contributes to epilepsy, a common neurological disorder characterized by recurrent seizures. Local overexpression of arachidonate lipoxygenase 3 (ALOXE3), a key enzyme for arachidonic acid (AA) metabolic pathway, alleviates seizure severities. However, the relationship between the ALOXE3 gene mutation and epilepsy has not been reported until now. Here we firstly characterized the promoter of human ALOXE3 gene and found that the ALOXE3 promoter could drive luciferase gene expression in the human HEK-293 and SH-SY5Y cells. We then screened the ALOXE3 promoter region and all coding exons from those patients with Dravet syndrome and identified 5 variants c.-163T > C, c.-50C > G, c.-37G > A, c. + 228G > A and c. + 290G > T in the promoter region and one missense variant c.1939A > G (p.I647 V) in the exon. Of these variants in the promoter region, only -50C > G was a novel variant located on the transcriptional factor NFII-I binding element. Luciferase reporter gene analyses indicated that the c.-50C > G could decrease gene expression by preventing the TFII-I's binding. In addition, the variant p.I647 V was conserved among all analyzed species and located within the ALOXE3 functional domain for catalyzing its substrate. In cultured cell lines, overexpression of ALOXE3 significantly decreased the cellular AA levels and overexpression of ALOXE3-I647 V could restore the AA levels, suggesting that the p.I647 V mutant led to a decrease in enzyme activity. Taken together, the present study proposes that the identified ALOXE3 variants potentially contribute to the AA-pathway-mediated epileptogenesis, which should provide a novel avenue for clinical diagnosis of epilepsy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Key Message: This report shows detailed characterization of LOX gene family in sorghum and provides new insight of sorghum LOX genes in genetic structure and their roles in plant response to infestation by sugarcane aphids. Lipoxygenases (LOXs) are monomeric, nonheme iron-containing dioxygenases that initiate the fatty acid oxidation pathway creating oxylipins and plant hormone jasmonate both have a key role in plant development and defense. To date, a comprehensive and systematic analysis of sorghum LOXs is still deficient. Thus, we performed a genome-wide analysis of the sorghum LOXs genome and identified nine LOXs genes. Detailed examination of protein sequences and phylogenetic analysis categorized the sorghum LOXs into two subclasses, 9-LOXs (SbLOX1, SbLOX3, SbLOX4, SbLOXm, and SbLOXo), 13-LOXs (SbLOX9, SbLOX5, and SbLOX2), and the unclassified SbLOX8. This classification was further supported by sequence similarity/identity matrix and subcellular localization analysis. The lipoxygenase domains, motifs, and vital amino acids were highly conserved in all sorghum LOX genes. In silico analysis of the promoter region of SbLOXs identified different hormones responsive cis-elements. Furthermore, to explore the roles of sorghum LOXs during sugarcane aphid feeding and exogenous MeJA application, expression analysis was conducted for all the eight LOXs in resistant (Tx2783) and susceptible (Tx7000) sorghum lines, respectively. As detailed in this report, the data generated from both genome-wide identification and expression analysis of lipoxygenase genes suggest the putative functions of two 13-LOXs (SbLOX9 and SbLOX5) and three 9-LOXs (SbLOX1, SbLOX3, and SbLOXo) in biosynthesis of jasmonic acid, green leaf volatiles and death acids, and all of them are involved in defense-related functions in plants. Furthermore, this report represents the first genome-wide analysis of the LOX gene family in sorghum, which will facilitate future studies to characterize the roles of each individual LOXs gene in aphid resistance and defense responses to other stresses.

Cold storage is widely used for delaying ripening and senescence; however, fruit aroma diminishes noticeably after long-term cold storage. The esters synthesized by the lipoxygenase (LOX) pathway are responsible for 'Nanguo' pear aroma. As methyl jasmonate (MeJA) is known to act on various fruit qualities, we investigated whether it acts via the LOX pathway in cold-stored 'Nanguo' pears. MeJA treatment increased the content of volatile esters and unsaturated fatty acids and the activities of alcohol acyltransferase, alcohol dehydrogenase, and LOX. It also up-regulated the expression of key genes (PuAAT, PuADH3, PuADH5, PuADH9, PuLOX1, and PuLOX3) in the LOX pathway and that of transcription factors (PuMYB21-like, PuMYB108-like, PuWRKY61, PuWRKY72, and PuWRKY31), whose genes were differentially expressed in preliminary transcriptome analysis. Therefore, considering its effects on LOX pathway-related genes and transcription factors, MeJA may be useful in preventing cold-storage-induced decline in ester biosynthesis, aroma, and consequently the quality of cold-stored 'Nanguo' pears., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This paper is focused on eicosanoid signaling in insect immunology. We begin with eicosanoid biosynthesis through the actions of phospholipase A 2 , responsible for hydrolyzing the C18 polyunsaturated fatty acid, linoleic acid (18:2n-6), from cellular phospholipids, which is subsequently converted into arachidonic acid (AA; 20:4n-6) via elongases and desaturases. The synthesized AA is then oxygenated into one of three groups of eicosanoids, prostaglandins (PGs), epoxyeicosatrienoic acids (EETs) and lipoxygenase products. We mark the distinction between mammalian cyclooxygenases and insect peroxynectins, both of which convert AA into PGs. One PG, PGI 2 (also called prostacyclin), is newly discovered in insects, as a negative regulator of immune reactions and a positive signal in juvenile development. Two new elements of insect PG biology are a PG dehydrogenase and a PG reductase, both of which enact necessary PG catabolism. EETs, which are produced from AA via cytochrome P450s, also act in immune signaling, acting as pro-inflammatory signals. Eicosanoids signal a wide range of cellular immune reactions to infections, invasions and wounding, including nodulation, cell spreading, hemocyte migration and releasing prophenoloxidase from oenocytoids, a class of lepidopteran hemocytes. We briefly review the relatively scant knowledge on insect PG receptors and note PGs also act in gut immunity and in humoral immunity. Detailed new information on PG actions in mosquito immunity against the malarial agent, Plasmodium berghei , has recently emerged and we treat this exciting new work. The new findings on eicosanoid actions in insect immunity have emerged from a very broad range of research at the genetic, cellular and organismal levels, all taking place at the international level.

After 300 million years of evolution, the first land-living mammals reentered the marine environment some 50 million years ago. The driving forces for this dramatic lifestyle change are still a matter of discussion but the struggle for food resources and the opportunity to escape predators probably contributed. Reentering the oceans requires metabolic adaption putting evolutionary pressure on a number of genes. To explore whether eicosanoid signaling has been part of this adaptive response, we first explored whether the genomes of marine mammals involve functional genes encoding for key enzymes of eicosanoid biosynthesis. Cyclooxygenase (COX) and lipoxygenase (ALOX) genes are present in the genome of all marine mammals tested. Interestingly, ALOX12B, which has been implicated in skin development of land-living mammals, is lacking in whales and dolphins and genes encoding for its sister enzyme (ALOXE3) involve premature stop codons and/or frameshifting point mutations, which interrupt the open reading frames. ALOX15 orthologs have been detected in all marine mammals, and the recombinant enzymes exhibit similar catalytic properties as those of land-living species. All marine mammals express arachidonic acid 12-lipoxygenating ALOX15 orthologs, and these data are consistent with the Evolutionary Hypothesis of ALOX15 specificity. These enzymes exhibit membrane oxygenase activity and introduction of big amino acids at the triad positions altered the reaction specificity in favor of arachidonic acid 15-lipoxygenation. Thus, the ALOX15 orthologs of marine mammals follow the Triad concept explaining their catalytic specificity., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)