Plant Lipoxygenases Research Articles

Fluctuations of an exposed π-helix involved in lipoxygenase substrate recognition.

The second helix in lipoxygenases adapts to permit substrate access to the active site, but details of this process are varied and poorly understood. We therefore examined the dynamics of helix 2 in solutions of spin-labeled soybean lipoxygenase-1 and spin relaxation at 60 K of the spin-labels by catalytic iron. Helix 2 in soybean lipoxygenase structures is surface-exposed and contains one turn of π-helix, centrally located. A site-directed spin-label scan of 18 of the 21 helix 2 residues, and electron paramagnetic resonance, showed that the π-helical segment became unusually mobile, on a nanosecond time scale, under conditions favoring substrate binding (pH 9 and lipid addition), while segments before and after had relatively unchanged dynamics. Backbone dynamics of residues in the π-helical segment appeared to be correlated, at pH 9. Samples also were frozen to examine the polarity and proticity of the local environments, the effect of the local environment on intrinsic relaxation, and dipolar relaxation by two symmetries of catalytic iron. The average hyperfine tensor component, Azz, of four π-helix residues decreased by 1.75 G, with an increase in pH from 7 to 9, while it remained unaffected for nearby buried residues. Power saturation data suggested the change in polarity specific to the π-helix altered the intrinsic relaxation rates. Different symmetries of iron contributed to distance-dependent magnetic relaxation. We interpret these data to mean that a π-helix in the second helix of plant lipoxygenases is highly dynamic and is the site where lipid chains penetrate to inner helices that outline the substrate pocket.

Hydrogen Tunneling in a Prokaryotic Lipoxygenase

A bacterial lipoxygenase (LOX) shows a deuterium kinetic isotope effect (KIE) that is similar in magnitude and temperature dependence to the very large KIE of eukaryotic LOXs. This occurs despite the evolutionary distance, an ∼25% smaller catalytic domain, and an increase in Ea of ∼11 kcal/mol. Site-specific mutagenesis leads to a protein variant with an Ea similar to that of the prototypic plant LOX, providing possible insight into the origin of evolutionary differences. These findings, which extend the phenomenon of hydrogen tunneling to a prokaryotic LOX, are discussed in the context of a role for protein size and/or flexibility in enzymatic hydrogen tunneling.

Plant growth promoting rhizobacteria (PGPR) and entomopathogenic fungus bioformulation enhance the expression of defense enzymes and pathogenesis-related proteins in groundnut plants against leafminer insect and collar rot pathogen

The bioformulation of Pseudomonas fluorescens (Pf1 and TDK1) and Beauveria bassiana (B2) strains was evaluated individually and in combinations with and without chitin for their efficacy against leafminer insect and collar rot disease and the effect of the interaction between Pseudomonas, Beauveria and groundnut leafminer insect and collar rot pathogen in the expression of defense enzymes and pathogenesis-related proteins (PR-proteins) in groundnut. Among the various bioformulations, B2 + TDK1 + Pf1 (amended with or without chitin) formulation significantly reduced the incidence of leafminer and collar rot disease when compared to untreated control. A significant increase in the enzymatic activity of phenylalanine ammonia-lyase, peroxidase, polyphenol oxidase, chitinase, β-1,3-glucanase, superoxide dismutase, catalase, lipoxygenase, and phenolics in groundnut plants treated with B2 + TDK1 + Pf1 bioformulation (amended with or without chitin) and challenge inoculated with Aproaerema modicella and Sclerotium rolfsii. Native gel electrophoresis also revealed the expression of more isoforms of pathogenesis-related proteins and other defense enzymes viz., polyphenol oxidase and superoxide dismutase in plants treated with B2 + TDK1 + Pf1 mixture challenged with A. modicella and S. rolfsii. The present study reveals that sustained and timely induction and accumulation of these defense enzymes and PR-proteins enhance the resistance in groundnut against leafminer insect and collar rot disease.

Cloning and expression of three lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli

Three genes homologous to plant lipoxygenase genes were identified from the EST libraries of Marchantia polymorpha, in order to clarify the function of LOXs in bryophytes. Full-length genes were isolated using 5′- and 3′-RACE methods and named MpLOX1, MpLOX2, and MpLOX3, respectively. To investigate the enzymatic activities of liverwort LOXs, recombinant MpLOX1, MpLOX2, and MpLOX3 proteins were prepared from Escherichia coli cells expressing the corresponding gene. LC–MS/MS analyses and chiral column chromatography of their reaction products showed that MpLOX1 codes for 11S/15S-lipoxygenase against eicosapentaenoic acid and for 15S-lipoxygenase against arachidonic acid, and that MpLOX2 and MpLOX3 code for 15S-lipoxygenase against eicosapentaenoic and arachidonic acids. Phylogenetic analysis showed that the liverwort lipoxygenase genes separated from the ancestor of higher plants in the early stages of plant evolution. Quantification analyses suggested that arachidonic acid and eicosapentaenoic acid were preferred substrates. Furthermore, each liverwort lipoxygenase exhibited highest activity at pH 7.0 and dependency on Ca2+ ion in the oxygenation reaction.

LOX genes in blast fungus (Magnaporthe grisea) resistance in rice

Plant Lipoxygenases (LOX) are known to play major role in plant immunity by providing front-line defense against pathogen-induced injury. To verify this, we isolated a full-length OsLOX3 gene and also 12 OsLOX cDNA clones from Oryza sativa indica (cultivar Pusa Basmati 1). We have examined the role played by LOXs in plant development and during attack by blast pathogen Magnaporthe grisea. Gene expression, promoter region analysis, and biochemical and protein structure analysis of isolated OsLOX3 revealed significant homology with LOX super family. Protein sequence comparison of OsLOXs revealed high levels of homology when compared with japonica rice (up to100%) and Arabidopsis (up to 64%). Isolated LOX3 gene and 12 OsLOX cDNAs contained the catalytic LOX domains much required for oxygen binding and synthesis of oxylipins. Amino acid composition, protein secondary structure, and promoter region analysis (with abundance of motifs CGTCA and TGACG) support the role of OsLOX3 gene in providing resistance to diseases in rice plants. OsLOX3 gene expression analysis of root, shoot, flag leaf, and developing and mature seed revealed organ specific patterns during rice plant development and gave evidence to association between tissue location and physiological roles played by individual OsLOXs. Increased defense activity of oxylipins was observed as demonstrated by PCR amplification of OsLOX3 gene and upon inoculation with virulent strains of M. grisea and ectopic application of methyl jasmonate in the injured leaf tissue in adult rice plants.

Lead tolerance and physiological adaptation mechanism in roots of accumulating and non-accumulating ecotypes of Sedum alfredii

Lead (Pb) accumulation in soils affects plants primarily through their root systems. The aim of this study was to investigate early symptoms of the loss of membrane integrity and lipid peroxidation in root tissues and physiological adaptation mechanism to Pb in accumulating ecotypes (AE) and non-accumulating ecotypes (NAE) of Sedum alfredii under Pb stress in hydroponics. Histochemical in situ analyses, fluorescence imaging, and normal physiological analysis were used in this study. Pb accumulation in roots of both AE and NAE increased linearly with increasing Pb levels (0-200 μM), and a significant difference between both ecotypes was noted. Both loss of plasma membrane integrity and lipid peroxidation in root tissues became serious with increasing Pb levels, maximum tolerable Pb level was 25 and 100 μM for NAE and AE, respectively. Pb supplied at a toxic level caused a burst of reactive oxygen species (ROS) in root cells in both ecotypes. However, the root cells of AE had inherently higher activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), and lipoxygenase (LOX) in control plants, and the induction response of these antioxidant enzymes occurred at lower Pb level in AE than NAE. AE plants maintained higher ascorbic acid and H(2)O(2) concentrations in root cells than NAE when exposed to different Pb levels, and Pb induced more increase in dehydroascorbate (DHA), catalase (CAT), and ascorbate peroxidase (APX) in AE than NAE roots. Results indicate that histochemical in situ analyses of root cell death and lipid peroxidation under Pb short-term stress was sensitive, reliable, and fast. Higher tolerance in roots of accumulating ecotype under Pb stress did depend on effective free oxygen scavenging by making complex function of both constitutively higher activities and sensitive induction of key antioxidant enzymes in root cells of S. alfredii.

Green leaf divinyl ether synthase: Gene detection, molecular cloning and identification of a unique CYP74B subfamily member

Enzymes of the CYP74 family (P450 superfamily) play a key role in the plant lipoxygenase signalling cascade. Recently we detected a pathogen inducible divinyl ether synthase (DES) in flax leaves [Chechetkin, Blufard, Hamberg, Grechkin, 2008]. This prompted us to examine the CYP74 genes in the flax leaf transcriptome. Since the flax genome is not sequenced, we used the PCR approach with degenerate primers related to the conserved domains of selected CYP74 genes; this revealed several CYP74 transcripts in flax leaves. One transcript belongs to the previously described allene oxide synthase ( LuAOS, CYP74A, GenBank ID: U00428.1). Another one contains the ORF (1473 bp) of an unknown CYP74B16 gene. Three more nearly identical sequences, including one expressed pseudogene, were also identified. The recombinant CYP74B16 protein expressed in Escherichia coli had 491 amino acid residues and MW of 56 kDa. The preferred substrate of this enzyme is the 13-hydroperoxide of α-linolenic acid, and the reaction product was identified by mass spectrometry, NMR and UV spectroscopy as the divinyl ether (9 Z,11 E)-12-[(1′ Z,3′ Z)-hexadienyloxy]-9,11-dodecadienoic acid, (ω5 Z)-etherolenic acid. All previously known CYP74B subfamily enzymes are hydroperoxide lyases. The novel flax enzyme CYP74B16 (LuDES) is an unprecedented DES member of the CYP74B subfamily.

Cloning, characterization and expression analysis of a 9-lipoxygenase gene in Gladiolus hybridus

Plant lipoxygenases (LOXs) is a functionally diverse class of dioxygenases involved in multiple physiological processes such as growth, senescence, and stress-related responses. Previously, based on the analysis of LOX1 activity and corm formation, it was depicted that LOX1 increased jasmonic acid (JA) synthesis and induced enlargement at top of stolon branches, forming cormels in Gladiolus hybridus. Here, LOX gene ( GhLOX1) in full length was isolated from developing corms in G. hybridus. Sequence analysis showed that GhLOX1 was a novel LOX1 gene encoding 9-LOX. Transient expression analysis determined GhLOX1 to be a cytoplasm protein. Real time RT-PCR showed that the mRNA level of GhLOX1 gene correlated positively with LOX activity and was highest in cormels. GhLOX1 mRNA level, LOX activity and endogenous methyl jasmonate (MJ) content in corms increased steadily with a MJ gradient from 0.1 mmol/L to 0.5 mmol/L. Conversely, GhLOX1 mRNA level, LOX activity, and endogenous MJ content in corms steadily decreased with the increasing concentration of Salicylhydroxamic acid (SHAM). At the same time, we observed that MJ and SHAM treatments effected both LOX1 mRNA level and LOX activity, leading to promotion and inhibition of corm formation and enlargement, respectively. These data reveal that GhLOX1 is a novel LOX1 gene encoding a cytoplasm 9-LOX protein and its expression would contribute to LOX activity which plays an important role in corm formation and enlargement. In general, GhLOX1 is an ideal candidate for the promotion of corm formation and enlargement by gene manipulation in G. hybridus.

Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and α-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy α-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.

Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies

Lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family.

Genome-wide identification, phylogeny and expression analysis of the lipoxygenase gene family in cucumber

Plant lipoxygenase (LOX) is involved in growth and developmental control processes, through the biosynthesis of regulatory molecules and defense responses to pathogens, wounding and stress. To date, few LOX proteins and little tissue expression profiling have been reported in detail for cucumber (Cucumis sativus L.). Recent completion of the cucumber genome sequence now permits genome-wide analysis of the LOX gene family in cucumber as well as comparison with LOX in Arabidopsis and rice. We identified 23 candidate LOX genes in the cucumber genome; phylogenetic analysis indicated that these LOX members cluster into two groups, designated types 1 and 2, as expected from previous studies. Sequence analysis showed that five binding sites of iron, including two consensus histidines in the LOX domain, are highly conserved in the cucumber LOX proteins. Analysis of chromosomal localization and genome distribution suggested that tandem duplication and/or polyploidal duplication contributed to the expansion of the cucumber LOX gene family. Based on intron/exon structure analysis, only a few of the extant intron patterns existed in the ancestor of monocots and eudicots. Expression data showed widespread distribution of the cucumber LOX gene family within plant tissues, suggesting that they perform different functions in different tissues.

Biochemical Characterization, Kinetic Analysis and Molecular Modeling of Recombinant Vegetative Lipoxygenases from Soybean

Plants in general have multiple lipoxygenase isoforms that exhibit distinct biochemical attributes and discrete cellular and sub-cellular localization patterns. This suggests that plant lipoxygenases are multifunctional and contribute to several biochemical and physiological processes during plant growth and development. Soybeans have at least eight isoforms and the three that are localized to the seed (L-1; L-2; L-3) have been characterized extensively and L-1 serves as a model. The vegetative organs, however, have five additional isoforms that are not well characterized at present. To better understand the cellular function of these vegetative lipoxygenases, kinetic parameters and pH profiles for the recombinant VLXs were determined for three free fatty acids above and below the corresponding critical micelle concentrations, including free fatty acids and the triglyceride trilinolein. The kinetic data using trilinolein as a substrate from two isoforms, VLX-C & VLX-D, supports a potential role for VLX-C & -D in the metabolism of lipids in soybean leaves as suggested by the significantly higher kcat/km and turnover of lipid substrates for these two isoforms. In addition, structural studies indicate clear differences exist between VLX-A, -B & -E and VLX-C & -D in the exposed loops of the ?-barrel, O2 cavity and entry site of the subcavity IIa. Therefore, based on distinct patterns of activity and structure, the existence of functional subgroups among the five VLX isoforms was confirmed.

Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with the transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using several deuterated AAs, including 13- pro-( S) d-AA, 13,13-d 2-AA and 10, 10, 13,13-d 4-AA. The primary KIE for steady-state cyclooxygenase catalysis, D k cat , ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9–2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes a slow formation of a pentadienyl AA radical and a rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of plant lipoxygenases indicates that PGHS and lipoxygenases have very different mechanisms of hydrogen transfer.

Molecular analysis of lipoxygenase ( LOX) genes in common wheat and phylogenetic investigation of LOX proteins from model and crop plants

Lipoxygenase (LOX) proteins constitute an important class of lipid-hydrolyzing enzymes in cellular organisms. Here, we report a molecular analysis of LOX genes in common wheat ( Triticum aestivum L.) and a more comprehensive phylogenetic investigation of the LOX proteins from higher plants. The full-length nucleotide sequences of two LOX genes ( TaLOX1 and TaLOX2) from common wheat were isolated. TaLOX1 and TaLOX2 were assigned to the short arm of chromosome 4D and the long arm of chromosome 5D, respectively. TaLOX1 and TaLOX2 were both expressed in the developing grains of two common wheat varieties, indicating that they may contribute to the total LOX activity in common wheat seeds. A more extensive phylogenetic analysis conducted with 143 unique LOXs from model and crop plants revealed that plant LOXs could be classified into two subfamilies, which correlated with the absence (subfamily I) or presence (subfamily II) of a predicted chloroplast targeting peptide in the deduced LOX proteins. Our work provides new information on the LOX genes in common wheat and the phylogenetic relationships of higher plant LOXs, which may aid further functional and evolutionary studies of plant LOXs.

Hexadecanoid pathway in plants: Lipoxygenase dioxygenation of (7Z,10Z,13Z)-hexadecatrienoic acid

7,10,13-Hexadecatrienoic acid (16:3) is abundant in many plant species. However, its metabolism through the lipoxygenase pathway is not sufficiently understood. The goal of present work was to investigate the oxygenation of 16:3 by different plant lipoxygenases and to study the occurrence of oxygenated derivatives of 16:3 in plant seedlings. The recombinant maize 9-lipoxygenase specifically converted 16:3 into (7S)-hydroperoxide. Identification of this novel oxylipin was substantiated by data of GC-MS, LC-MS/MS, 1H-NMR, and 2D-COSY as well as by deuterium labeling from [(2)H(6)]16:3. Soybean lipoxygenase 1 produced 91% (11S)-hydroperoxide and 6% racemic 14-hydroperoxide. Recombinant soybean lipoxygenase 2 (specifically oxidizing linoleate into 13-hydroperoxide) lacked any specificity towards 16:3. Lipoxygenase 2 produced 7-, 8-, 10-, 11-, 13-, and 14-hydroperoxides of 16:3, as well as a significant amount of bis-allylic 9-hydroperoxide. Seedlings of several examined plant species possessed free hydroxy derivatives of 16:3 (HHTs), as well as their ethyl esters. Interestingly, HHTs occur not only in "16:3 plants", but also in typical "18:3 plants" like pea and soybean seedlings.

Differential expression pattern of an acidic 9/13-lipoxygenase in flower opening and senescence and in leaf response to phloem feeders in the tea plant

BackgroundLipoxygenase (LOXs) is a large family of plant enzymes that catalyse the hydroperoxidation of free polyunsaturated fatty acids into diverse biologically active compounds, collectively named phyto-oxylipins. Although multiple isoforms of LOXs have been identified in a wide range of annual herbaceous plants, the genes encoding these enzymes in perennial woody plants have not received as much attention. In Camellia sinensis (L.) O. Kuntze, no LOX gene of any type has been isolated, and its possible role in tea plant development, senescence, and defence reaction remains unknown. The present study describes the isolation, characterization, and expression of the first tea plant LOX isoform, namely CsLOX1, and seeks to clarify the pattern of its expression in the plant's defence response as well as in flower opening and senescence.ResultsBased on amino acid sequence similarity to plant LOXs, a LOX was identified in tea plant and named CsLOX1, which encodes a polypeptide comprising 861 amino acids and has a molecular mass of 97.8 kDa. Heterologous expression in yeast analysis showed that CsLOX1 protein conferred a dual positional specificity since it released both C-9 and C-13 oxidized products in equal proportion and hence was named 9/13-CsLOX1. The purified recombinant CsLOX1 protein exhibited optimum catalytic activity at pH 3.6 and 25°C. Real-time quantitative PCR analysis showed that CsLOX1 transcripts were detected predominantly in flowers, up-regulated during petal senescence, and down-regulated during flower bud opening. In leaves, the gene was up-regulated following injury or when treated with methyl jasmonate (MeJA), but salicylic acid (SA) did not induce such response. The gene was also rapidly and highly induced following feeding by the tea green leafhopper Empoasca vitis, whereas feeding by the tea aphid Toxoptera aurantii resulted in a pattern of alternating induction and suppression.ConclusionsAnalysis of the isolation and expression of the LOX gene in tea plant indicates that the acidic CsLOX1 together with its primary and end products plays an important role in regulating cell death related to flower senescence and the JA-related defensive reaction of the plant to phloem-feeders.

Identification of the lipoxygenase gene family from Vitis vinifera and biochemical characterisation of two 13-lipoxygenases expressed in grape berries of Sauvignon Blanc

Lipoxygenases (LOXs) are a group of non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids (PUFAs) and lipids, and initiate the formation of biologically active compounds known as oxylipins. Several plant oxylipins comprise important flavours and aromas in food and beverages. Analysis of the grape (Vitis vinifera L.) genome revealed that the grape LOX family consists of 18 individual members. Phylogenetic analysis places all except one of the identified grape LOXs into either a type II 13-LOX cluster or the type I 9-LOX cluster. Four LOX genes (VvLOXA, VvLOXO, VvLOXC, VvLOXD), representative of the major LOX groupings observed in the phylogenetic analyses, were selected for analysis of patterns of transcript abundance in berry tissues. VvLOXA and VvLOXO represent putative 13-LOXs, while VvLOXC represents a putative 9-LOX. VvLOXD represents a unique LOX that differs significantly from other characterised plant LOXs. All four LOXs exhibited a complex pattern of gene expression. Across all developmental stages, VvLOXA was the most abundant LOX and was expressed predominantly in berry skins. The expression pattern of VvLOXC and -D are more evenly distributed between seeds, pulp and skin, while VvLOXO is mostly expressed in the seed. Mechanical wounding and infection of berries with Botrytis cinerea Pers.: Fr resulted in rapid accumulation of VvLOXC and -O transcripts. VvLOXA expression decreased in diseased berries. Biochemical analysis of VvLOXA and -O recombinant proteins confirmed that these LOX genes encode functional 13-LOXs that exhibit different pH and temperature optima. Both enzymes showed activity with linoleic, linolenic and arachidonic acids.

Specificity of oxidation of linoleic acid homologs by plant lipoxygenases

The lipoxygenase-catalyzed oxidation of linoleic acid homologs was studied. While the linoleic acid oxidation by maize 9-lipoxygenase (9-LO) specifically produced (9S)-hydroperoxide, the dioxygenation of (11Z,14Z)-eicosadienoic (20:2) and (13Z,16Z)-docosadienoic (22:2) acids by the same enzyme lacked regio- and stereospecificity. The oxidation of 20:2 and 22:2 by 9-LO afforded low yields of racemic 11-, 12-, 14-, and 15-hydroperoxides or 13- and 17-hydroperoxides, respectively. Soybean 13-lipoxygenase-1 (13-LO) specifically oxidized 20:2, 22:2, and linoleate into (omega6S)-hydroperoxides. Dioxygenation of (9Z,12Z)-hexadecadienoic acid (16:2) by both 9-LO and 13-LO occurred specifically, affording (9S)- and (13S)-hydroperoxides, respectively. The data are consistent with the "pocket theory of lipoxygenase catalysis" (i.e. with the penetration of a substrate into the active center with the methyl end first). Our findings also demonstrate that the distance between carboxyl group and double bonds substantially determines the positioning of substrates within the active site.

A lipoxygenase with dual positional specificity is expressed in olives ( Olea europaea L.) during ripening

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered.

Measurement of Lipid Hydroperoxides by the Ferric-Xylenol Orange Method (2) Application to Lipoxygenase Assay

The ferric-xylenol orange (FOX) method has been used for quantification of hydroperoxides by measuring the colored ferric-xylenol orange (XO/Fe3+) product with a peak absorbance at 560 nm. We recently reported a modified FOX method, the sensitivity of which was increased in the presence of membranous phosphatidylcholine (PC) by forming a XO/Fe3+-PC complex with a peak absorbance at 610 nm. Lipoxygenases (LOXs) and their metabolites have been implicated in a wide range of disease states. We applied our newly developed FOX method to the assay of human 15-lipoxygenase-2 (15-LOX-2) and soybean lipoxygenase (SLOX) as typical animal and plant lipoxygenases, respectively. The amounts of 15-S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (15-HPETE) produced by 15-LOX-2 measured by UV-absorption at 237 nm attributed to the conjugated diene, coincided with the results of our FOX method measuring absorbance at 610 nm. The 15-HPETE production time courses measured by the two methods also correlated well. SLOX rapidly oxidized unesterified linoleic acids (LA) and slowly esterified fatty acids in egg yolk PC (EYPC). Availability of EYPC was increased if the membrane structure was moderately disturbed by MeOH and Triton X-100, but LA oxidation was readily decreased by them. These results indicate that our method is useful for lipoxygenase assay. Furthermore, our method was applicable to assaying the inhibitory effect of 5,8,11,14-eicosatetraynoic acid (ETYA) on SLOX activity using LA and EYPC as substrates. The inhibition dose-dependency of ETYA was almost the same in the LA and EYPC systems, although the enzyme concentrations differed by a factor of 1,000, suggesting that ETYA functioned as a competitive inhibitor. These results indicate that our method may be useful as a screen for the identification of novel inhibitors of lipoxygenases.