Biosynthesis of the Maresin Intermediate, 13S,14S-Epoxy-DHA, by Human 15-Lipoxygenase and 12-Lipoxygenase and Its Regulation through Negative Allosteric Modulators.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater kcat/KM value (14.0 ± 0.8 s-1 µM-1) than that of h15-LOX-1 (0.36 ± 0.08 s-1 µM-1) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller kcat/KM value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s-1 µM-1, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (Kd = 6.0 µM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 3.5 µM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPAω-3) (Kd = 4.0 µM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (Kd = 3.5 µM) and 14S-HDPAω-3 (Kd = 4.0 µM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

[Screening of Organisms Producing Inhibitors of 15-Lipoxygenase Among Micromycetes.]

The effects of extracts from the mycelium of Lecanicilium lecaniiNo.169, Beauveria fellina No.7 and Beauveria bassianaNo.15 on the activity of 15-lpoxygenase (15-LO) recovered from rat reticulocytes was investigated. The activity of 15-LO was determined by oxidation of linolic acid. The extract from the mycelium of the fungal complex was shown to inhibit 15-LO (IC50 of 12 mcg/ml). The inhibitory effect of the combined extract on 15-LO was due to the substances recovered from Lecanicilium lecanii No.169. The extract fractions responsible for the activity were determined and the compounds containing the fractions were identified. They proved to be 10 - 4-hydroxybenzoic acid and 4-hydroxybenzyl alcohol and genistein, a flavonoid from fraction 11. The possible role of the inhibitory effect of the compounds on 15-LO in the antiatherosclerotic activity of the fungal extract is discussed.

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.

Development of a fluorescent intensity assay amenable for high-throughput screening for determining 15-lipoxygenase activity.

15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z' value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC(50) determinations.

Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30-80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2.

Structure-activity relationship studies of flavonoids as potent inhibitors of human platelet 12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2.

Human lipoxygenase (hLO) isozymes have been implicated in a number of disease states and have attracted much attention with respect to their inhibition. One class of inhibitors, the flavonoids, have been shown to be potent lipoxygenase inhibitors but their study has been restricted to those compounds found in nature, which have limited structural variability. We have therefore carried out a comprehensive study to determine the structural requirements for flavonoid potency and selectivity against platelet 12-hLO, reticulocyte 15-hLO-1, and prostate epithelial 15-hLO-2. We conclude from this study that catechols are essential for high potency, that isoflavones and isoflavonones tend to select against 12-hLO, that isoflavons tend to select against 15-hLO-1, but few flavonoids target 15-hLO-2.

Baicalein is a potent in vitro inhibitor against both reticulocyte 15-human and platelet 12-human lipoxygenases.

Lipoxygenases (LO) have been implicated in asthma, immune disorders, and various cancers and as a consequence, there is great interest in isolating selective LO isozyme inhibitors. Currently, there is much use of baicalein as a selective human platelet 12-LO (12-hLO) inhibitor, however, our current steady-state inhibition data indicate that baicalein is not selective against 12-hLO versus human reticulocyte 15-LO-1 (15-hLO-1) (15/12=1.3), in vitro. However, in the presence of detergents baicalein is slightly more selective (15/12=7) as seen by the steady-state inhibition kinetics, which may imply greater selectivity in a cell-based assay but has yet to be proven. The mechanism of baicalein inhibition of 15-hLO-1 is reductive, which molecular modeling suggests is through direct binding of the catecholic moiety of baicalein to the iron. A structurally related flavonoid, apigenin, is not reductive, however, molecular modeling suggests a hydrogen bond with Thr591 may account for its inhibitor potency.

Structural flexibility of the N-terminal beta-barrel domain of 15-lipoxygenase-1 probed by small angle X-ray scattering. Functional consequences for activity regulation and membrane binding.

Mammalian lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases (atherosclerosis, osteoporosis) with major health political importance. The crystal structures of two plant lipoxygenase isoforms have been solved and X-ray coordinates for an inhibitor complex of the rabbit 15-lipoxygenase-1 are also accessible. Here, we investigated the solution structure of the ligand-free rabbit 15-lipoxygenase-1 by small angle X-ray scattering. From the scattering profiles we modeled the solution structure of the enzyme using two independent ab initio approaches. Preliminary experiments indicated that at low protein concentrations (<1mg/ml) and at 10 degrees C the enzyme is present as hydrated monomer. Superposition of the high resolution crystal structure and our low resolution model of the solution structure revealed two major differences. (i) Although the two models are almost perfectly superimposed in the region of the catalytic domain the solution structure is stretched out in the region of the N-terminal beta-barrel domain and exhibits a bigger molecular volume. (ii) There is a central bending of the enzyme molecule in the solution structure, which does not show up in the crystal structure. Both structural peculiarities may be explained by a high degree of motional freedom of the N-terminal beta-barrel domain in aqueous solutions. This interdomain movement may be of functional importance for regulation of the catalytic activity and membrane binding.

Inhibition studies of soybean and human 15-lipoxygenases with long-chain alkenyl sulfate substrates.

Lipoxygenases are currently potential targets for therapies against asthma, artherosceloris, and cancer. Recently, inhibition studies on both soybean (SLO) and human lipoxygenase (15-HLO) revealed the presence of an allosteric site that binds both substrate, linoleic acid, and inhibitors; oleic acid (OA) and oleyl sulfate (OS). OS (K(D) approximately 0.6 microM) is a approximately 30-fold more potent inhibitor than OA (K(D) approximately 20 microM) due to the increased ionic strength of the sulfate moiety. To further investigate the role of the sulfate moiety on lipoxygenase function, SLO and 15-HLO were assayed against several fatty sulfate substrates (linoleyl sulfate (LS), cis-11,14-eicosadienoyl sulfate, and arachidonyl sulfate). The results demonstrate that SLO catalyzes all three fatty sulfate substrates and is not inhibited, indicating a binding selectivity of LS for the catalytic site and OS for the allosteric site. The 15-HLO, however, manifests parabolic inhibition kinetics with increasing substrate concentration, and it is irreversibly inhibited by these fatty sulfate substrates at high concentrations. The inhibition can be stopped, however, by the addition of detergent to the fatty sulfate mixture prior to the addition of 15-HLO. These results, combined with the modeling of the kinetic data, indicate that the inhibition of 15-HLO is due to a substrate aggregate. These substrate aggregates, however, do not inhibit SLO and could present a novel mode of inhibition for 15-HLO.

Defining the arachidonic acid binding site of human 15-lipoxygenase. Molecular modeling and mutagenesis.

Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg402) and an aromatic amino acid residue (Phe414) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.

Role of lipid hydroperoxides in the activation of 15-lipoxygenase.

We have used stopped-flow rapid reaction methods, employing both fluorescence and absorbance monitoring, together with HPLC analysis of the products to study the activation of soybean 15-lipoxygenase by 13(S)-hydroperoxy-9, 11(E,Z)-octadecadienoic acid (13-HPOD). When lipoxygenase is mixed with an equimolar concentration of 13-HPOD, the enzyme undergoes a rapid change in fluorescence. The rate of the change of fluorescence is dependent on the concentration of the 13-HPOD (k = 6.7 x 10(6) M-1 s-1) and is accompanied by activation of the enzyme. The fluorescence change is not accompanied by any change in the UV absorbance of the 13-HPOD, suggesting no loss of the conjugated diene during enzyme activation, and HPLC analysis of the products of the reaction confirms that the 13-HPOD can be recovered unchanged following this reaction. In the presence of an inhibitor (BWA4C, a hydroxamate inhibitor) that reduces the active-site iron, the 13-HPOD and the inhibitor are destroyed in a peroxidase-like reaction. On the basis of these observations we propose that 13-HPOD binds to the enzyme and facilitates activation of the enzyme, possibly through the formation of a protein radical, and that the 13-HPOD is not changed chemically in this process.

Lipoxygenase activity in rat dermis and epidermis: partial purification and characterization.

Lipoxygenase (LOX) activity in epidermis and dermis was distributed among microsomal and cytosolic fractions. The main products of polyunsaturated fatty acid metabolism were 12-hydroperoxy-cis-5,8,14, trans-10-eicosatetraenoic acid (12-HPETE), 15-hydroperoxy-cis-5,8,11, trans-13-eicosatetraenoic acid (15-HPETE) and 13-hydroxy-cis-9, trans-11-octadecadienoic acid (13-HOD). Enzyme activities were isolated from rat dermis and epidermis by ammonium sulphate precipitation, hydrophobic chromatography and gel filtration. In the dermis, activity was found at a molecular mass of 68 kDa, a pI of 4.6 and a Km of 50 microM. This activity was inhibited by known LOX inhibitors. The main reaction products indicated that this was 15-LOX. In the epidermis, activity was found in a fraction with a molecular mass of 68 kDa, a pI of 4.6 and a Km of 80 microM. Activity was inhibited by known LOX inhibitors whereas the reaction products indicated that this was 12-LOX. LOX activity in rat skin may involve one enzyme with dual regional specificities or may comprise two different enzymes.

Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.

Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes. By comparing conserved differences among sequences of 12- and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified. Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase. An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase. Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculo-virus-insect cell culture system. This variant was purified to > 90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase. The variant enzyme has no change in its apparent KM for arachidonic acid and a minor (3-fold) change in its Vmax. For linoleic acid, the variant has no change in its KM and a 10-fold reduction in its Vmax, as expected for an enzyme performing predominantly 12-lipoxygenation. The results are consistent with a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme. Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation.

Overexpression, purification and characterization of human recombinant 15-lipoxygenase.

Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.

Bacterial expression, purification and partial characterization of recombinant rabbit reticulocyte 15-lipoxygenase.

The recombinant rabbit reticulocyte 15-lipoxygenase has been expressed in E. coli with a yield of about 50-70 micrograms pure lipoxygenase protein per 1 of liquid culture. The enzyme has been purified to apparent homogeneity from the bacteria lysis supernatant by ammonium sulfate precipitation, and two consecutive steps of anion exchange chromatography on a Mono Q column. As the native enzyme the recombinant lipoxygenase has a molecular mass of 75 kDa, an isoelectric point of 5.5 and oxygenates both linoleic acid (formation of 13S-hydroperoxy-9Z,13E-octadecadienoic acid) and arachidonic acid. With the latter substrate it exhibits a dual positional specificity (formation of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetranoic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid in a ratio of 12:1). Furthermore, the enzyme is capable of oxygenating biomembranes, as indicated by HPLC analysis of esterified oxygenated polyenoic fatty acids.

Rapid purification of rabbit reticulocyte lipoxygenase for electron paramagnetic spectroscopy characterization of the non-heme iron.

An efficient three-step purification technique has been developed for the reticulocyte 15-lipoxygenase from rabbit. Ammonium sulfate fractionated reticulocyte lysate was purified by size exclusion chromatography and preparative scale isoelectric focusing. The entire procedure was complete in less than eight hours and was carried out in batches which typically yielded 10 mg of purified enzyme. The identity and purity of the enzyme were evaluated by N-terminal sequencing, sodium dodecylsulfate polyacrylamide gel electrophoresis and specific activity determinations. The enzyme contained approximately one g-atom iron per mole of protein. The iron was present in an electron paramagnetic spectroscopy (EPR) silent, presumably high spin iron(II), form in the isolated enzyme. Treatment with one equivalent of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid resulted in the appearance of an EPR signal around g6.

Purification of two forms of arachidonate 15-lipoxygenase from human leukocytes.

Two different proteins with arachidonate 15-lipoxygenase activity have been purified to near homogeneity from human leukocytes. Both have the same molecular mass (74 kDa) on SDS/PAGE and appear to be equally active with three different fatty acid substrates. The N-terminal amino acid sequences of both forms were identical to the sequence of human reticulocyte 15-lipoxygenase [Sigal, E., Craik, C.S., Highland, E., Grunberger, D., Costello, L.L., Dixon, R.A.F. & Nadel, J.A. (1988) Biochem. Biophys. Res. Commun. 157, 457-464]. The two forms of 15-lipoxygenase could be clearly separated by cation-exchange chromatography. Of particular interest, the relative amounts of the two forms differed markedly between leukocytes obtained from normal donors and leukocytes from an individual with eosinophilia.

The airway epithelium and arachidonic acid 15-lipoxygenase.

Pulmonary epithelial cells may be primarily responsible for initiating or regulating inflammatory responses in the airways, in part by releasing chemical mediators. Among the most potent mediators of inflammation are the lipoxygenase metabolites of arachidonic acid, including the leukotrienes and other mono and dihydroxyeicosatetraenoic acids (HETES). The human airway epithelium contains significant 15-lipoxygenase activity. Although some biologic functions of 15-lipoxygenase metabolites are known, further understanding of the role of this enzyme in the airway requires localization in tissue and studies of expression, regulation, and biologic activity. Towards these aims, we purified and characterized 15-lipoxygenase from eosinophil-enriched leukocytes. First, we studied cofactors that may be involved in regulating enzymatic activity. Second, we isolated to homogeneity, for the first time, human 15-lipoxygenase. This led to the determination of the N-terminal amino acid sequence and the discovery of homology among various mammalian lipoxygenases. Finally, we utilized this structural information to isolate a cDNA that encodes for human 15-lipoxygenase. The availability of a clone will permit studies of expression and the development of antibodies for tissue localization. Further research using molecular and antibody probes is expected to increase our understanding of the biologic roles of 15-lipoxygenase in airway epithelium.

Purification and crystallization of 15-lipoxygenase from rabbit reticulocytes.

We report a new purification of rabbit reticulocyte 15-lipoxygenase that has resulted in the first crystallization of a mammalian lipoxygenase. The enzyme was purified to homogeneity (greater than 98% pure by SDS-PAGE) using high pressure liquid chromatography on hydrophobic-interaction, hydroxyapatite and cation-exchange columns. Crystals were grown by the vapor diffusion method from concentrated solutions of the protein in sodium phosphate buffer, pH 7.0. The hexagonal, rod-shaped crystals were on average 0.09 mm x 0.09 mm x 0.4 mm, with approximate unit cell dimensions of a = b = 260 A, c = 145 A. The crystals diffract to 5 A resolution.