Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.     

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme–substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.     

Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes

5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (> 95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity.     

Stereocontrol of arachidonic acid oxygenation by vertebrate lipoxygenases: newly cloned zebrafish lipoxygenase 1 does not follow the Ala-versus-Gly concept

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.     

A rapid miniature thin layer chromatography system for analysis of prostaglandins an lipoxygenase products

We have developed a miniature thin layer chromatography system for rapidly identifying the major arachidonate metabolites in a radiolabeled form elaborated by cells or tissues. This system separates the total spectrum of cyclooxygenase products and several of the most commonly found lipoxygenase pathway metabolites, while retaining the fine resolution of larger-scale and more time-consuming procedures. It requires less than one hour for extraction of metabolites, chromatography, and counting.     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium1

Bovine corneal epithelium contains arachidonate 12- and 15-lipoxygenase activity, while human corneal epithelium contains only 15-lipoxygenase activity. Our purpose was to identify the corneal 12- and 15-lipoxygenase isozymes. We used cDNA cloning to isolate the amino acid coding nucleotide sequences of two bovine lipoxygenases. The translated sequence of one lipoxygenase was 82% identical with human 15-lipoxygenase type 2 and 75% identical with mouse 8-lipoxygenase, whereas the other translated nucleotide sequence was 87% identical with human 12-lipoxygenase of the platelet type. Expression of 15-lipoxygenase type 2 and platelet type 12-lipoxygenase mRNAs were detected by Northern analysis. In addition to these two lipoxygenases, 12-lipoxygenase of leukocyte (tracheal) type was detected by polymerase chain reaction (PCR), sequencing, and Northern analysis. Finally, PCR and sequencing suggested that human corneal epithelium contains 15-lipoxygenase types 1 and 2.     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

The pH-dependence of the Escherichia coli RNase HII-catalysed reaction suggests that an active site carboxylate group participates directly in catalysis

RNase HII specifically catalyses the hydrolysis of phosphate diester linkages contained within the RNA portion of DNA/RNA hybrids. The catalytic parameters of the enzyme derived from Escherichia coli BL21 have been measured using 5'-fluorescent oligodeoxynucleotide substrates containing embedded ribonucleotides. The products of the reaction and the chemistry of phosphate diester hydrolysis were assigned unequivocally using mass spectrometry. The pH-dependence of the catalytic parameters was measured under conditions of optimal magnesium ion concentration. The logarithm of the turnover number of the enzyme increases steeply with pH until a pH-independent region is reached close to neutrality. The slope of the pH-dependent region is 2, indicating that the catalytically proficient form of RNase HII is di-anionic. The pH-dependence of log 1/K(M) is a sigmoidal curve reaching a maximal value at higher pH, suggesting deprotonation of a residue stabilises substrate binding. Possible mechanisms for the RNase HII-catalysed reaction consistent with the pH-dependent behaviour of the enzyme are discussed. The active sites of RNase H enzymes contain a cluster of four strictly conserved carboxylate groups. Together, the data suggest a requirement for ionisation of an active site carboxylic acid for metal ion binding or correct positioning of metal ion(s) in the enzyme-substrate complex and a role for a second active site carboxylate in general base catalysis.     

LC/ESR/MS study of pH-dependent radical generation from 15-LOX-catalyzed DPA peroxidation

Docosapentaenoic acid (DPA) is a unique fatty acid that exists in two isomeric forms (n-3 and n-6), which differ in their physiological behaviors. DPA can undergo free radical-mediated peroxidation via lipoxygenase (LOX). 15-LOX, one of the LOX isomers, has received much attention in cancer research because of its very different expression level in normal tissues compared to tumors and some bioactive fatty acid metabolites modulating the tumorigenic pathways in cancer. However, the mechanism linking 15-LOX, DPA metabolites, and their bioactivities is still unclear, and the free radicals generated in DPA peroxidation have never been characterized. In this study, we have studied radicals formed from both soybean and human cellular (PC3-15LOS cells) 15-LOX-catalyzed peroxidation of DPAs at various pH's using a combination of LC/ESR/MS with the spin trapping technique. We observed a total of three carbon-centered radicals formed in 15-LOX-DPA (n-3) stemming from its 7-, 17-, and 20-hydroperoxides, whereas only one formed from 17-hydroperoxide in DPA (n-6). A change in the reaction pH from 8.5 (15-LOX enzyme optimum) to 7.4 (physiological) and to 6.5 (tumor, acidic) not only decreased the total radical formation but also altered the preferred site of oxygenation. This pH-dependent alteration of radical formation and oxygenation pattern may have significant implications and provide a basis for our ongoing investigations of LOXs as well as fatty acids in cancer biology.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB

All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.     

The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier

This review covers the background to discovery of the two key lipoxygenases (LOX) involved in epidermal barrier function, 12R-LOX and eLOX3, and our current views on their functioning. In the outer epidermis, their consecutive actions oxidize linoleic acid esterified in ω-hydroxy-ceramide to a hepoxilin-related derivative. The relevant background to hepoxilin and trioxilin biochemistry is briefly reviewed. We outline the evidence that linoleate in the ceramide is the natural substrate of the two LOX enzymes and our proposal for its importance in construction of the epidermal water barrier. Our hypothesis is that the oxidation promotes hydrolysis of the oxidized linoleate moiety from the ceramide. The resulting free ω-hydroxyl of the ω-hydroxyceramide is covalently bound to proteins on the surface of the corneocytes to form the corneocyte lipid envelope, a key barrier component. Understanding the role of the LOX enzymes and their hepoxilin products should provide rational approaches to ameliorative therapy for a number of the congenital ichthyoses involving compromised barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Identification of Novel Decenoic Acids in Heated Butter

Novel decenoic acids such as (E)-4-decenoic acid and (E)- and (Z)-5-,6-decenoic acid were detected as minor components in heated butter using GC and GC/MS. The formation mechanism of these novel decenoic acids is discussed on the basis of the result of the reaction of δ-decalactone with active clay in a model experiment.