Plant Lipoxygenases Research Articles

Oxylipin biosynthesis via an unprecedented 16-hydroperoxide lyase pathway in green tissues of cucumber (Cucumis sativus L.) plants

The plant lipoxygenase cascade is a source of various regulatory oxylipins that play a role in cell signalling, stress adaptation, and immune response. Recently, we detected an unprecedented 16(S)-lipoxygenase, CsLOX3, in the leaves and fruit pericarp of cucumber (Cucumis sativus L.). In the present work, an array of products biosynthesized through the conversions of α-linolenic acid 16-hydroperoxide (16-HPOT) was detected. Firstly, a prominent 15-hydroxy-9,12-pentadecadienoic acid (Me/TMS) was detected, the product of hydroperoxide lyase (HPL) chain cleavage of 16-HPOT and further reduction of aldehyde 15-oxo-9,12-pentadecadienoic acid to alcohol. Besides, the presence of dicarboxylic acid, 3,6-pentadecadiene-1,15-dioic acid, was deduced from the detection of its catalytic hydrogenation product, pentadecane-1,15-dioic acid. Finally, 12,15-dihydroxypentadecanoic acid (Me/TMS) was detected amongst the hydrogenated products, thus indicating the presence of the parent 12,15-dihydroxy-9,13-pentadecadienoic acid. To confirm the proposed HPL chain cleavage, the 16(S)-HPOT was prepared and incubated with the recombinant cucumber HPL CYP74B6 enzyme. The CYP74B6 possessed high activity towards 16-HPOT. Chain cleavage yields the (9Z,12Z)-15-oxo-9,12-pentadecadienoic acid, undergoing a spontaneous isomerization into (9Z,13E)-15-oxo-9,13-pentadecadienoic acid. Thus, the cucumber plants as well as the recombinant cucumber HPL CYP74B6 possessed unprecedented 16-HPL activity, cleaving 16-HPOT into a C15 fragment, 15-oxo-9,12-pentadecadienoic acid, and a complementary volatile C3 fragment, propionic aldehyde. The 16-LOX/16-HPL route of oxylipin biosynthesis presents a novel facet of the plant LOX pathway.

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.

Identification of the Thermal Activation Network in Human 15-Lipoxygenase-2: Divergence from Plant Orthologs and Its Relationship to Hydrogen Tunneling Activation Barriers.

The oxidation of polyunsaturated fatty acids by lipoxygenases (LOXs) is initiated by a C-H cleavage step in which the hydrogen atom is transferred quantum mechanically (i.e., via tunneling). In these reactions, protein thermal motions facilitate the conversion of ground-state enzyme-substrate complexes to tunneling-ready configurations and are thus important for transferring energy from the solvent to the active site for the activation of catalysis. In this report, we employed temperature-dependent hydrogen-deuterium exchange mass spectrometry (TDHDX-MS) to identify catalytically linked, thermally activated peptides in a representative animal LOX, human epithelial 15-LOX-2. TDHDX-MS of wild-type 15-LOX-2 was compared to two active site mutations that retain structural stability but have increased activation energies (Ea) of catalysis. The Ea value of one variant, V427L, is implicated to arise from suboptimal substrate positioning by increased active-site side chain rotamer dynamics, as determined by X-ray crystallography and ensemble refinement. The resolved thermal network from the comparative Eas of TDHDX-MS between wild-type and V426A is localized along the front face of the 15-LOX-2 catalytic domain. The network contains a clustering of isoleucine, leucine, and valine side chains within the helical peptides. This thermal network of 15-LOX-2 is different in location, area, and backbone structure compared to a model plant lipoxygenase from soybean that exhibits a low Ea value of catalysis compared to the human ortholog. The presented data provide insights into the divergence of thermally activated protein motions in plant and animal LOXs and their relationships to the enthalpic barriers for facilitating hydrogen tunneling.

CRISPR/Cas9-mediated lipoxygenase gene-editing in yellow pea leads to major changes in fatty acid and flavor profiles.

Although pulses are nutritious foods containing high amounts of protein, fiber and phytochemicals, their consumption and use in the food industry have been limited due to the formation of unappealing flavors/aromas described as beany, green, and grassy. Lipoxygenase (LOX) enzymes are prevalent among pulse seeds, and their activity can lead to the formation of specific volatile organic compounds (VOCs) from certain polyunsaturated fatty acids (PUFAs). As a widespread issue in legumes, including soybean, these VOCs have been linked to certain unappealing taste perception of foods containing processed pulse seeds. To address this problem in pea and as proof of principle to promote the wider use of pulses, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) construct was designed to create null alleles (knockouts) of PsLOX2 which had been implicated in the generation of VOCs in peas. Successful CRISPR/Cas9-mediated LOX gene editing of stable transgenic pea lines (TGP) was confirmed by DNA sequencing of the wild type (WT) and TGP pslox2 mutant lines. These lines were also assessed for LOX activity, PUFA levels, and VOCs. Compared to WT peas, the TGP lines showed a significant reduction (p < 0.05) in LOX activity and in the concentration of key VOCs, including hexanal, 2-hexenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, 1-octen-3-ol, octanal, (E)-2-octenal (E,E)-2,4-nonadienal and furan-2-pentyl. The content of two essential PUFAs, linoleic and α-linolenic acids, the known substrates of LOX in plants, was higher in TGP flours, indicating the efficacy of the CRISPR-mediated gene editing in minimizing their oxidation and the further modification of PUFAs and their products. The collection of VOCs from the headspace of ground pea seeds, using a portable eNose also distinguished the TGP and WT lines. Multiple regression analysis showed that LOX activity correlated with the two VOCs, heptanal and (E,E)-2,4-heptadienal in pea flours. Partial Least Squares Regression (PLS-R) plot for selected PUFAs, VOCs, and sensor responses in WT and TGP lines showed distinct clusters for WT and TGP lines. Together this data demonstrates the utility of CRISPR mediated mutagenesis of PsLOX2 to quickly improve aroma and fatty acid (FA) profiles of pea seeds of an elite Canadian variety.

Isolation and partial characterization of the lipoxygenase gene in black pepper (Piper nigrum L)

Black pepper is a spice that have an economic importance in the world by its wide application in industry and by medicinal properties. State of Pará is one of the largest producers of these spices, but the production of this species is committed by fusariosis, that causes root rot in plant. Study of molecular biology is important by support breeding improvement of black pepper. In this work we draw the primers Lox3R and Lox3F to amplify a larger fragment of the gene lipoxygenase (Lox) in black pepper. The amplified fragment using primers drawn was sequenced. The sequence isolated has 770 nucleotides that encode 258 amino acids. This sequence was characterized by comparison in biological databases and using computational programs. The analysis with BlastX showed that sequence isolated has high similarity with lipoxygenase proteins of Persea americana, Parasponia andersonii e Vitis vinífera. We verified that Lox of black pepper has the PLAT/ LH2 domain of plant lipoxygenase related proteins. Its description in black pepper is essential to clarify the molecular mechanisms of response of the plant to the Fungus and understand its paper in activation of the defense response, once the gene Lox is activated in plant to signal its defense in a possible attack against pathogen and may be precursors of metabolic regulators.

Structural and functional evaluation mammalian and plant lipoxygenases upon association with nanodics as membrane mimetics

Lipoxygenases (LOX) are a family lipid oxygenating enzymes that can generate bioactive lipids of clinical relevance from polyunsaturated fatty acids. Most LOXs display a Ca2+-dependent association with membranes for their activity. Nanodiscs (ND) are stable self-assembled discoidal fragments of lipid bilayers that can mimic the plasma membrane. In this study, we evaluated the association of mammalian 15-LOXs (ALOX15 and ALOX15B) and soybean LOX-1 with NDs (LOX-ND), their enzymatic activities and inhibition. Mammalian LOXs associated with NDs showed better retention of enzymatic function compared to soybean LOX-1. Treatment of both LOX-NDs and free enzymes with the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) showed an approximately 5-fold more effective inhibition of the enzymes associated with NDs compared to the free form. NDs are easy to generate membrane mimics that can be used as an effective tool to determine enzymatic function and inhibition of membrane associated proteins.

The Responses of the Lipoxygenase Gene Family to Salt and Drought Stress in Foxtail Millet (Setaria italica).

Plant lipoxygenases (LOXs), a kind of non-heme iron-containing dioxygenases, participate plant physiological activities (especially in response to biotic and abiotic stresses) through oxidizing various lipids. However, there was few investigations on LOXs in foxtail millet (Setaria italica). In this study, we identified the LOX gene family in foxtail millet, and divided the total 12 members into three sub-families on the basis of their phylogenetic relationships. Under salt and drought stress, LOX genes showed different expression patterns. Among them, only SiLOX7 showed up-regulated expression in Yugu1 (YG1) and Qinhuang2 (QH2), two stress-tolerant varieties, indicating that SiLOX7 may play an important role in responses to abiotic stress. Our research provides a basis for further investigation of the role of LOX genes in the adaptation to abiotic stresses and other possible biological functions in foxtail millet.

Optimization of Hydroperoxide Lyase Production for Recombinant Lipoxygenase Pathway Cascade Application

Cis-3-hexenal and its more stable isomer, trans-2-hexenal, are highly valued chemicals used in the food and perfume industries. They are produced by the plant lipoxygenase pathway, where two enzymes, lipoxygenase (LOX) and hydroperoxide lyase (HPL), are involved. However, the application of this pathway is limited, especially due to the instability of HPL. This enzyme belongs to the cytochrome P450 enzyme family and needs heme as a prosthetic group. Its synthesis must be effectively performed by a host organism in order to produce an active protein. In this work, Pseudomonas aeruginosa LOX was expressed in Escherichia coli BL21(DE3), and whole cells were used for the synthesis of 13(S)-hydroperoxy-(Z,E,Z)-9,11,15-octadecatrienoic acid (13-HPOT) as a substrate for HPL. Expression of Psidium guajava HPL was carried out by recombinant E. coli JM109(DE3) in autoinduction media, and the influence of the addition of heme precursors δ-ALA and FeII+ was studied. Specific activity of whole cells expressing HPL was measured by the direct use of a synthesized 13-HPOT solution (2.94 mM of total hydroperoxides, 75.35% of 13-HPOT (2.22 mM)) and increased 2.6-fold (from 61.78 U·mg−1 to 159.95 U·mg−1) with the addition of 1 mM FeII+ to the autoinduction media. Productivity and activity were further enhanced by an increase in the expression temperature, and a total of 3.30·105 U·dm−3 of culture media was produced in the optimized process.

Exploring Two Streptomyces Species to Control Rhizoctonia solani in Tomato

Streptomyces species are effective biocontrol agents toward many plant pathogens. These microorganisms are well known for producing secondary metabolites, promoting plant growth and inducing plant defense mechanisms. In this study, the ability of tomato root-colonizing Streptomyces strains to trigger the resistance against Rhizoctonia solani (J.G. Kühn) AG4 was investigated. For this goal, we evaluated the pattern of LOXB and PAL1 genes expression changes upon pathogen inoculation in primed tomato plants. The results revealed that Streptomyces globisporous (Krasil’nikov) strain F8 and S. praecox (Millard and Burr) strain R7 were able to enhance the expression of lipoxygenase and phenylalanine ammonia lyase in tomato plants. This finding suggests that Streptomyces strains F8 and R7 may trigger jasmonic acid and phenyl propanoid signaling pathways in plants, therefore, resulting an induced defense status in tomatoes against R. solani. Biochemical characterization of these Streptomyces strains showed that they were strong producers of siderophores. S. praecox strain R7 produced siderophores of hyderoxamate and catechol types and S. globisporous strain F8 produced a phenolic siderophore. Moreover, they also produced protease while only the S. praecox strain R7 was able to produce amylase. Taken together, these results indicate that S. globisporous strain F8 and S. praecox strain R7 promote plant growth and reduces disease and hence are suitable for future in depth and field studies with the aim to attain appropriate biocontrol agents to protect tomatoes against R. solani.

Loss of ZmLIPOXYGENASE4 Decreases Fusarium verticillioides Resistance in Maize Seedlings.

Fusarium verticillioides is one of the most relevant fungal species in maize responsible for ear, stalk and seedling rot, as well as the fumonisin contamination of kernels. Plant lipoxygenases (LOX) synthesize oxylipins that play a crucial role in the regulation of defense mechanisms against pathogens and influence the outcome of pathogenesis. To better uncover the role of these signaling molecules in maize resistance against F. verticillioides, the functional characterization of the 9-LOX gene, ZmLOX4, was carried out in this study by employing mutants carrying Mu insertions in this gene (named as UFMulox4). In this regard, the genotyping of five UFMulox4 identified the mutant UFMu10924 as the only one having an insertion in the coding region of the gene. The impact of ZmLOX4 mutagenesis on kernel defense against F. verticillioides and fumonisin accumulation were investigated, resulting in an increased fungal susceptibility compared to the inbred lines W22 and Tzi18. Moreover, the expression of most of the genes involved in the LOX, jasmonic acid (JA) and green leaf volatiles (GLV) pathways, as well as LOX enzymatic activity, decreased or were unaffected by fungal inoculation in the mutant UFMu10924. These results confirm the strategic role of ZmLOX4 in controlling defense against F. verticillioides and its influence on the expression of several LOX, JA and GLV genes.

Approving the biocontrol method of potato wilt caused by Ralstonia solanacearum (Smith) using Enterobacter cloacae PS14 and Trichoderma asperellum T34

This study aimed to evaluate the efficiency of Enterobacter cloacae PS14 and Trichoderma asperellum T34 in the control of potato wilt, caused by Ralstonia solanacearum (Smith), under greenhouse and field conditions. In vitro, the endophyte E. cloacae PS14 caused the highest reduction of the pathogen growth among 7 screened bacteria. It produced an inhibition zone as 16.9 mm compared to a specific antibiotic (20.0 mm). E. cloacae PS14 was selected as an effective antagonistic bacterium to be compared to T. asperellum strain T34 for reduction of the disease as well as increasing the crop yield of potato plants. Both E. cloacae and T. asperellum reduced the disease severity up to 10.7–26.5%, respectively, under greenhouse and up to 26.6–36.6%, respectively, under field conditions. The results approved that both E. cloacae and T. asperellum increased the yield of the crop by 20.44–40.96%, respectively. Their mode of action was indicated by suppression of the pathogen as well as induction of plant systemic resistance. The induction of systemic resistance was confirmed by increasing the total phenol and salicylic acid contents as well as increasing the activities of peroxidase, lipoxygenase, and polyphenol oxidase in potato plants than the healthy or only infected plants. Production of siderophore, indole-3-acetic acid (0.577–0.884 μM), hydrogen cyanide (2.34–3.61 μg/ml), and salicylic acid (0.436–1.488 μg/ml) was confirmed by E. cloacae PS14 and T. asperellum T34, respectively, in vitro. The study recommends the new strain E. cloacae PS14, as new endophytic effective bacteria, in the control of R. solanacearum causing the potato wilt disease.

Plant Lipoxygenases and Their Role in Plant Physiology

Several enzymes play a key role in plant growth and development. Among these, lipoxygenases family of enzymes are important. Lipoxygenases are a ubiquitous family of non-heme iron enzymes widely distributed in plants, initiate hydroperoxidation of polyunsaturated fatty acids containing cis, cis-1,4-pentadiene moieties, and produce phytooxylipins. Oxylipins including green leaf volatiles and hormone jasmonic acid play a significant role in physiological processes, seed germination, fruit ripening, and senescence. Detailed studies have shown that LOX and its products accumulate transiently upon developmental or environmental stimuli. LOXs play a key role in defense responses against biotic and abiotic environmental stresses such as fungi, bacteria, virus, nematodes, birds, insects, and rodents. As a result, these enzymes are the natural topics of study when scientists investigate plant response to stresses. In this review, we have concisely focused on the analysis of new information concerning plant LOXs and their role on the physiology of plants.

Oxygenation reactions catalyzed by the F557V mutant of soybean lipoxygenase-1: Evidence for two orientations of substrate binding

Plant lipoxygenases oxygenate linoleic acid to produce 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPOD) or 9-hydroperoxy-10E,12Z-octadecadienoic acid (9(S)-HPOD). The manner in which these enzymes bind substrates and the mechanisms by which they control regiospecificity are uncertain. Hornung et al. (Proc. Natl. Acad. Sci. USA96 (1999) 4192–4197) have identified an important residue, corresponding to phe-557 in soybean lipoxygenase-1 (SBLO-1). These authors proposed that large residues in this position favored binding of linoleate with the carboxylate group near the surface of the enzyme (tail-first binding), resulting in formation of 13(S)-HPOD. They also proposed that smaller residues in this position facilitate binding of linoleate in a head-first manner with its carboxylate group interacting with a conserved arginine residue (arg-707 in SBLO-1), which leads to 9(S)-HPOD. In the present work, we have tested these proposals on SBLO-1. The F557V mutant produced 33% 9-HPOD (S:R = 87:13) from linoleic acid at pH 7.5, compared with 8% for the wild-type enzyme and 12% with the F557V,R707L double mutant. Experiments with 11(S)-deuteriolinoleic acid indicated that the 9(S)-HPOD produced by the F557V mutant involves removal of hydrogen from the pro-R position on C-11 of linoleic acid, as expected if 9(S)-HPOD results from binding in an orientation that is inverted relative to that leading to 13(S)-HPOD. The product distributions obtained by oxygenation of 10Z,13Z-nonadecadienoic acid and arachidonic acid by the F557V mutant support the hypothesis that ω6 oxygenation results from tail-first binding and ω10 oxygenation from head-first binding. The results demonstrate that the regiospecificity of SBLO-1 can be altered by a mutation that facilitates an alternative mode of substrate binding and adds to the body of evidence that 13(S)-HPOD arises from tail-first binding.

Antimicrobial Activity of Geometric Isomers of Etherolenic Acid-the Products of Plant Lipoxygenase Cascade.

Data on the influence of the double bond geometry on the antimicrobial properties of different isomers of etherolenic acid against phytopathogenic bacteria are presented. (ω5Z)-Etherolenic acid possesses bactericidal properties against Xanthomonas campestris ssp. vesicatoria, Pseudomonas syringae ssp. tomato, Pectobacterium atrosepticum SCRI1043; the etherolenic and (11Z)-etherolenic acids possess only bacteriostatic properties.

Resposta da soja, com e sem supressão de lipoxigenases, às injúrias mecânicas e do percevejo-marrom

The effect of the genetic elimination of lipoxygenases in soybean plants aiming to reduce beany flavor was verified in the induction of defense, when the plant is infested with E. heros herbivore or mechanically injured by comparison with plants with regular genotype. The enzymatic activity of the lipoxygenase pool in the regular genotype was higher in plants with mechanical injuries in the grains, if compared to the control without injuries after 8 hours. In the triple null genotype, the enzyme activity was lower in plants infested with the herbivore. The results indicate the importance of the lipoxygenase isoenzymes presence in soybean response to herbivore attack and mechanical injury, and, additionally, the importance of the enzyme presence in signaling systemic injuries, which may be the reason soybeans are more susceptible to herbivore infestation.

HPLC Analysis and Biochemical Characterization of LOX from Eschscholtzia californica Cham.

Background: Plant lipoxygenases (LOXs, EC 1.13.11.12) are involved in lipid degradation, regulation of growth and development, senescence, and defence reactions. LOX represents the starting enzyme of the octadecanoid pathway. The aim of the work was to purify LOX from California poppy (Eschscholtzia californica Cham.), to determine its biochemical properties and to identify and quantify the products of LOX reaction with unsaturated fatty acids. Methods: LOX from California poppy seedlings was purified by hydrophobic chromatography (Phenyl-Sepharose CL-4B) and by ion-exchange chromatography (Q-Sepharose). The isolated LOX was incubated with linoleic acid used as a substrate. The HPLC experiments were performed with the Agilent Technologies 1050 series HPLC system. For the preparative separation of a mixture of hydroxy fatty acids from the sample matrix, the RP-HPLC method was used (column 120-5 Nucleosil C18). Then, the NP-HPLC analysis (separation, identification, and determination) of hydroxy fatty acid isomers was carried out on a Zorbax Rx-SIL column. Results: The purified LOX indicates the presence of a nontraditional plant enzyme with dual positional specificity (a ratio of 9- and 13-hydroperoxide products 1:1), a relative molecular mass of 85 kDa, a pH optimum of 6.5, an increasing activity stimulation by CaCl2 till 2 mM, and a high substrate reactivity to linoleic acid with kinetic values of KM 2.6 mM and Vmax 3.14 μM/min/mg. Conclusions: For the first time, the LOX from California poppy seedlings was partially purified and the biochemical properties of the enzyme were analyzed. A dual positional specificity of the LOX found from California poppy seedlings is in agreement with the results obtained for LOXs isolated from other Papaveraceaes. A 1:1 ratio of 9-/13-HODE is attractive for the simultaneous investigation of both biotic stress responses (indicated by the 9-HODE marker) and the biosynthesis of jasmonic acid and jasmonates (indicated by the 13-HODE marker).

Using Mutagenesis and Synthetic Substrates to Explore Regiochemical Control by Soybean‐Lipoxygenase‐1

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Plant lipoxygenases oxygenate linoleic acid to produce either 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD), 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD) or a mixture of both. The factors determining the position of oxygen incorporation are not well understood. Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197) identified one critical residue, corresponding to phenylalanine‐557 in soybean lipoxygenase‐1 (SBLO‐1). These workers observed that lipoxygenases such as SBLO‐1 with phenylalanine or histidine in this position generally produce primarily 13‐HPOD, whereas those with valine or threonine produce primarily 9‐HPOD. In the present work, the F557V mutant of SBLO‐1 has been prepared. Consistent with the trend noted by Hornung et al., this mutant produces 30–40% 9‐HPOD from linoleic acid at pH 7.5, compared with less than 10% for the wild‐type enzyme. Hornung et al. suggested that having a smaller residue in position 557 might enable linoleic acid to bind in a head‐first manner with the carboxylate group of the substrate interacting with arginine‐707. Consistent with head‐first binding, the 9‐HPOD formed by the F557V mutant has predominantly (86%) the S‐configuration at carbon‐9. To further test the model, we have investigated the action of SBLO‐1 on linoleoyldiethylamine (LDMA), which has been previously shown to be a good substrate for wild‐type SBLO‐1 at pH 7.0, where the dimethylamino group of LDMA is expected to be protonated and positively charged, which would disfavor interaction with arginine‐707. With this substrate, the amount of oxygenation at carbon‐9 is greater than observed with linoleic acid. This result argues against the hypothesis that oxygenation at carbon‐9 arises from binding of the polar end of the substrate near arginine‐707. Surprisingly, the F557A mutant produces only 9% 9‐HPOD from linoleic acid. This result indicates that the amount of 9‐oxygenation does not always correlate with the size of the residue at position‐557. In sum, the results confirm the importance of residue 557 in determining the regiochemistry of the products, but the structural basis for these effects remains to be elucidated.Support or Funding InformationNSF CHE‐1213262

A functional role identified for conserved charged residues at the active site entrance of lipoxygenase with double specificity

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of free polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and α-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13-hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the “double specificity” character of these LOXs. In this study, we used the olive LOX1 with double specificity to investigate the implication of the charged residues R265, R268, and K283 in the orientation of the substrate into the active site. These residues are present in a conserved pattern around the entrance of the active site. Our results show that these residues are involved in the penetration of the substrate into the active site: this positive patch could capture the carboxylate end of the substrate, and then guide it into the active site. Due to its position on α2 helix, the residue K283 could have a more important role, its interaction with the substrate facilitating the motions of residues constituting the “cork of lipoxygenases” or the α2 helix, by disrupting putative hydrogen and ionic bonds.

Resistance to Fusarium verticillioides and fumonisin accumulation in maize inbred lines involves an earlier and enhanced expression of lipoxygenase (LOX) genes

Fusarium verticillioides causes ear rot in maize and contaminates the kernels with the fumonisin mycotoxins. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defence against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. The expression profiles of fifteen genes of the LOX pathway were studied in kernels of resistant and susceptible maize lines, grown in field condition, at 3, 7 and 14 days post inoculation (dpi) with F. verticillioides. Plant defence responses were correlated with the pathogen growth, the expression profiles of fungal FUM genes for fumonisin biosynthesis and fumonisin content in the kernels. The resistant genotype limited fungal growth and fumonisin accumulation between 7 and 14 dpi. Pathogen growth became exponential in the susceptible line after 7 dpi, in correspondence with massive transcription of FUM genes and fumonisins augmented exponentially at 14 dpi. LOX pathway genes resulted strongly induced after pathogen inoculation in the resistant line at 3 and 7 dpi, whilst in the susceptible line the induction was reduced or delayed at 14 dpi. In addition, all genes resulted overexpressed before infection in kernels of the resistant genotype already at 3 dpi. The results suggest that resistance in maize may depend on an earlier activation of LOX genes and genes for jasmonic acid biosynthesis.

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-(2)H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the (2)H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.