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Hartmut Kühn - One of the best experts on this subject based on the ideXlab platform.

  • Do Lipoxygenases occur in viruses?: Expression and characterization of a viral Lipoxygenase-like protein did not provide evidence for the existence of functional viral Lipoxygenases.
    Prostaglandins Leukotrienes and Essential Fatty Acids, 2018
    Co-Authors: Tatjana Gehring, Dagmar Heydeck, Agathe Niewienda, Katharina Janek, Hartmut Kühn

    Abstract Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and animals. In bacteria, these enzymes are rare and have been introduced via horizontal gene transfer. Since viruses function as horizontal gene transfer vectors and since Lipoxygenases may be helpful for releasing assembled virus particles from host cells we explored whether these enzymes may actually occur in viruses. For this purpose we developed a four-step in silico screening strategy and searching the publically available viral genomes for Lipoxygenase-like sequences we detected a single functional gene in the genome of a mimivirus infecting Acantamoeba polyphaga. The primary structure of this protein involved two putative metal ligand clusters but the recombinant enzyme did neither contain iron nor manganese. Most importantly, it did not exhibit Lipoxygenase activity. These data suggests that this viral Lipoxygenase-like sequence does not encode a functional Lipoxygenase and that these enzymes do not occur in viruses.

  • Specificity and inhibition by antioxidant of lipid peroxidation by Lipoxygenase: effects of substrate, Lipoxygenase and milieu
    International Congress Series, 2002
    Co-Authors: Noriko Noguchi, Hartmut Kühn, Hiromasa Yamashita, Etsuo Niki

    Abstract Although lipid peroxidation by Lipoxygenases usually results in a site-, stereo- and enantio-specific mechanism, it was found that the oxidation of phospholipids incorporated into the liposomal membrane by soybean Lipoxygenase, but not by rabbit reticulocyte 15-Lipoxygenase, was proceeded entirely by a nonspecific mechanism. The radical-scavenging antioxidants such as α-tocopherol did not inhibit specific oxidation; however, they did suppress the nonspecific oxidation.

  • Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes.
    Free Radical Biology and Medicine, 2002
    Co-Authors: Hartmut Kühn, Astrid Borchert

    For a long time lipid peroxidation has only been considered a deleterious process leading to disruption of biomembranes and thus, to cellular dysfunction. However, when restricted to a certain cellular compartment and tightly regulated, lipid peroxidation may have beneficial effects. Early on during evolution of living organisms special lipid peroxidizing enzymes, called Lipoxygenases, appeared and they have been conserved during phylogenesis of plants and animals. In fact, a diverse family of Lipoxygenase isoforms has evolved starting from a putative ancient precursor. As with other enzymes, Lipoxygenases are regulated on various levels of gene expression and there are endogenous antagonists controlling their cellular activity. Among the currently known mammalian Lipoxygenase isoforms only 12/15-Lipoxygenases are capable of directly oxygenating ester lipids even when they are bound to membranes and lipoproteins. Thus, these enzymes represent the pro-oxidative part in the cellular metabolism of complex hydroperoxy ester lipids. Its metabolic counterplayer, representing the antioxidative part, appears to be the phospholipid hydroperoxide glutathione peroxidase. This enzyme is unique among glutathione peroxidases because of its capability of reducing ester lipid hydroperoxides. Thus, 12/15-Lipoxygenase and phospholipid hydroperoxide glutathione peroxidase constitute a pair of antagonizing enzymes in the metabolism of hydroperoxy ester lipids, and a balanced regulation of the two proteins appears to be of major cell physiological importance. This review is aimed at summarizing the recent developments in the enzymology and molecular biology of 12/15-Lipoxygenase and phospholipid hydroperoxide glutathione peroxidase, with emphasis on cytokine-dependent regulation and their regulatory interplay.

  • Mammalian 15-Lipoxygenases
    Advances in Experimental Medicine and Biology, 1999
    Co-Authors: Hartmut Kühn, Sabine Borngräber

    Lipoxygenases are enzymes which dioxygenate polyunsaturated fatty acids to hydroperoxy derivatives. Although the mechanism of the enzyme catalysis is not entirely clear, it has been suggested that the Lipoxygenase reaction involves the formation of an enzyme-bound fatty acid radical which is formed via a stereoselective removal of a hydrogen from a doubly allylic methylene group.1 It should be stressed that there are alternative explanations of the reaction mechanism implicating an electron removal from a double bond forming a fatty acid cation and a subsequent abstraction of a proton which would require a strong basic residue at the active site of the enzyme.2 Assuming the radical mechanism, the Lipoxygenase reaction resembles that of the non-enzymatic lipid peroxidation. In principle, both reactions can be divided into three steps (Figure 1): i) hydrogen abstraction, ii) radical rearrangement and iii) oxygen insertion. During the Lipoxygenase reaction each of the three steps is enzyme-controlled which leads to a specific pattern of oxygenation products. If, for instance, more than one doubly allylic methylene is present in the substrate fatty acid, Lipoxygenases select one of them for initial hydrogen abstraction.3 In contrast, during non-enzymatic lipid peroxidation, hydrogen is removed from all doubly allylic methylenes. Similarly, the introduction of dioxygen proceeds stereoselectively in the case of the Lipoxygenase reaction, whereas a stereorandom oxygenation is observed for the non-enzymatic lipid peroxidation.

  • the diversity of the Lipoxygenase family many sequence data but little information on biological significance
    FEBS Letters, 1999
    Co-Authors: Hartmut Kühn, Bernd J Thiele

    Lipoxygenases form a family of lipid peroxidising enzymes, which oxygenate free and esterified polyenoic fatty acids to the corresponding hydroperoxy derivatives. They are widely distributed in both the plant and animal kingdoms. During the last couple of years more and more Lipoxygenase isoforms have been discovered but for most of them the biological significance remains unclear. This review attempts to classify the currently known mammalian Lipoxygenase isoforms and critically reviews the concepts for their biological importance.

Angeles Manresa - One of the best experts on this subject based on the ideXlab platform.

  • Crystallization of the Lipoxygenase of Pseudomonas aeruginosa 42A2, evolution and phylogenetic study of the subfamilies of the Lipoxygenases
    Co-Authors: Albert Garreta, Xavi Carpena, Montse Busquets, Carmen Fusté, Ignacio Fita, Angeles Manresa

    Lipoxygenases are non-heme iron enzymes essential in eukaryotes, where they catalyze the formation of the fatty acid hydroperoxides that are required by a large diversity of biological and pathological processes. In prokaryotes, most of them totally lacking in polyunsaturated fatty acids, the possible biological roles of Lipoxygenases have remained obscure. In this study, it is reported the crystallization of a Lipoxygenase of Pseudomonas aeruginosa (Pa_LOX), the first from a prokaryote. High resolution data has been acquired which is expected to yield structural clues to the questions adressed. Besides, a preliminar phylogenetic analysis Correspondence/Reprint request: Dr. Albert Garreta, Institute of Research in Biomedicine (IRB-Barcelona) and Institut de Biologia Molecular (IBMB-CSIC), Parc Cientific, Barcelona, Spain E-mail: Albert Garreta et al. 248 using 14 sequences has confirmed the existence of this subfamily of bacterial Lipoxygenases, on one side, and a greater diversity than in the corresponding eukaryotic ones, on the other. Finally, an evolutionary study of bacterial Lipoxygenases on the same set of Lipoxygenases, show a selection pressure of a basically purifying or neutral character except for a single aminoacid, which would have been selected after a positive selection event.

  • Bacterial Lipoxygenases, a new subfamily of enzymes? A phylogenetic approach
    Applied Microbiology and Biotechnology, 2013
    Co-Authors: Jhoanne Hansen, Albert Garreta, Maria Benincasa, M. Carmen Fusté, Montserrat Busquets, Angeles Manresa

    Lipoxygenases (EC. are a non-heme iron enzymes consisting of one polypeptide chain folded into two domains, the N-terminal domain and the catalytic moiety β-barrel domain. They catalyze the dioxygenation of 1Z,4Z-pentadiene moieties of polyunsaturated fatty acids obtaining hydroperoxy fatty acids. For years, the presence of Lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates, and fungi, but now, some Lipoxygenase sequences have been detected on prokaryotic organisms, changing the idea that Lipoxygenases are exclusively a eukaryotic affair. Lipoxygenases are involved in different types of reactions on eukaryote organisms where the biological role and the structural characteristics of these enzymes are well studied. However, these aspects of the bacterial Lipoxygenases have not yet been elucidated and are unknown. This revision discusses biochemical aspects, biological applications, and some characteristics of these enzymes and tries to determine the existence of a subfamily of bacterial Lipoxygenases in the context of the phylogeny of prokaryotic Lipoxygenases, supporting the results of phylogenetic analyzes with the comparison and discussion of structural information of the first prokaryotic Lipoxygenase crystallized and other eukaryotic Lipoxygenases structures.

Colin D. Funk - One of the best experts on this subject based on the ideXlab platform.

  • basal transepidermal water loss is increased in platelet type 12 Lipoxygenase deficient mice
    Journal of Investigative Dermatology, 1999
    Co-Authors: Eric N Johnson, Jyoti Virmani, Lillian B Nanney, John A Lawson, Colin D. Funk

    The roles of fatty acids in the skin have been under investigation since early reports of the phenotypic abnormalities of mice fed a diet deficient in essential fatty acids. Little is known about the functional significance of fatty acid metabolism by Lipoxygenases in epidermis. Here, we have examined the role of platelet-type 12-Lipoxygenase which converts arachidonic acid to the oxygenated metabolite 12-hydroperoxyeicosatetraenoic acid, in the skin using platelet-type 12-Lipoxygenase-deficient mice generated by gene targeting. Platelet-type 12-Lipoxygenase in wild-type mice was localized to the stratum granulosum by immunohistochemical analysis. Platelet-type 12-Lipoxygenase-deficient mice lacked immunodetectable platelet-type 12-Lipoxygenase in platelets and epidermis, appeared grossly normal, and exhibited an increase in basal transepidermal water loss without alteration in basal mitotic activity. Water loss and mitotic activity in mice with an acetone-disrupted membrane barrier were normal. No defect in ultrastructural properties or content of major fatty acids in dorsal skin or ear inflammation response was apparent in platelet-type 12-Lipoxygenase-deficient mice. These results indicate that the platelet-type 12-Lipoxygenase pathway in mice is partly responsible for normal permeability barrier function but the mechanism awaits further elucidation.

  • disruption of the 12 15 Lipoxygenase gene diminishes atherosclerosis in apo e deficient mice
    Journal of Clinical Investigation, 1999
    Co-Authors: Tillmann Cyrus, Joseph L Witztum, Daniel J Rader, Rajendra K Tangirala, Sergio Fazio, Macrae F Linton, Colin D. Funk

    Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This “oxidation hypothesis” has gained general acceptance in recent years, and evidence for the role of Lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed Lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-Lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-Lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E‐deficient mice (apo E ‐/‐ /L-12LO ‐/‐ ). In chow-fed 15-week-old apo E ‐/‐ /L-12LO ‐/‐ mice, the extent of lesions in whole-aorta en face preparations (198 ± 60 μm 2 ) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-Lipoxygenase‐expressing controls (apo E ‐/‐ /L-12LO +/+ ), which showed areas of lipid deposition (15,700 ± 2,688 μm 2 ) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E‐deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E ‐/‐ /L-12LO ‐/‐ mice. The combined data indicate a role for 12/15Lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

  • Lipoxygenases of Mice and Men
    Eicosanoids, 1996
    Co-Authors: Colin D. Funk

    Seven years have elapsed since the cloning of the cDNA for the first mammalian Lipoxygenase and the deduced primary structure (Matsumoto et al., 1988; Dixon et al., 1988). Here, a brief comparative overview of the molecular properties of human and mouse Lipoxygenases will be presented. Although each of the human Lipoxygenases were isolated and systematically characterized prior to the corresponding murine homologs it became necessary to evaluate their relatedness in view of our present targeted Lipoxygenase gene inactivation experiments in mice.

Ernst H. Oliw - One of the best experts on this subject based on the ideXlab platform.

  • Catalytic properties of recombinant manganese(III)-Lipoxygenase and acid-catalyzed hydrolysis to mini-manganese-Lipoxygenase
    Co-Authors: Mirela Cristea, Åke Engström, Ernst H. Oliw

    Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (Lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known Lipoxygenase with catalytic manganese (Mn-Lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.Mn-Lipoxygenase belongs to the Lipoxygenase gene family. Recombinant Mn-Lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-Lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe Lipoxygenases.Mn-Lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-Lipoxygenase (~70 kDa). The active form of Mn-Lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).Mn-Lipoxygenase is an R-Lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S Lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of Lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-Lipoxygenase and LDS might provide new means to combat these pathogens.

  • Cloning of the manganese Lipoxygenase gene reveals homology with the Lipoxygenase gene family
    FEBS Journal, 2002
    Co-Authors: Lena Hörnsten, Chao Su, Anne Osbourn, Ulf Hellman, Ernst H. Oliw

    Manganese Lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.7 kDa) could be aligned with mammalian and plant Lipoxygenases with 23-28% identity over 350-400 amino-acid residues of the catalytic domains. Lipoxygenases have one water molecule and five amino acids as Fe ligands. These are two histidine residues in the highly conserved 30 amino-acid sequence WLLAK-X15-H-X4-H-X3-E of alpha helix 9, one histidine and usually an asparaine residue in the sequence H-X3-N-X-G of alpha helix 18, and the carboxyl oxygen of the C-terminal isoleucine (or valine) residue. The homologous sequence of alpha helix 9 of Mn-Lipoxygenase [WLLAK-X14-H(294)-X3-H(297)-X3-E] contained two single-amino-acid gaps, but otherwise His294 and His297 aligned with the two His residues, which coordinate iron. Mn-Lipoxygenase [H(478)-X3-N(482)-X-G] could be aligned with the two metal ligands of alpha helix 18, and the C-terminal residue was Val618. We conclude that Mn-Lipoxygenase belongs to the Lipoxygenase gene family and that its unique biochemical properties might be related to structural differences in the metal centre and alpha helix 9 of Lipoxygenases rather than to the metal ligands.

  • studies of Lipoxygenases in the epithelium of cultured bovine cornea using an air interface model
    Experimental Eye Research, 2000
    Co-Authors: Maria Liminga, Ernst H. Oliw

    Epithelial Lipoxygenases of bovine cornea were investigated in organ culture models. Subcellular fractions of the epithelium were incubated with14C-labelled arachidonate and the metabolites were analysed. Bovine corneal epithelial cells contain 15-Lipoxygenase type 2 and 12-Lipoxygenases of the leukocyte and the platelet types. The 15-Lipoxygenase activity was prominent in the cytosolic fraction. Twelve- and 15-Lipoxygenases occurred in the microsomal fraction, where the 15-Lipoxygenase activity appeared to be favoured by low protein levels. The Lipoxygenase activities strongly declined within 24 hr when the cornea was covered with cell culture medium, but were maintained with high activity in an air interface organ culture model for at least 72 hr. Cultured corneas were studied in pairs in the air interface model under influence of inflammatory stimuli. The epithelial 15- and 12-Lipoxygenase activities were only slightly augmented by treatment with 12- O -tetradecanoyl-phorbol-13-acetate (10 μ, 8–72 hr), and remained unchanged after treatment with lipopolysaccharide (1–100 μ g ml−1, 8–72 hr) or UV irradiation (301 nm, 0.17 J cm−2; 8–24 hr). In some experiments, 5-Lipoxygenase activity was detectable, as judged from liquid chromatography-mass spectrometry and chiral chromatography. Reverse transcription-polymerase chain reaction and Northern blot analysis were therefore used to identify mRNA of 5-Lipoxygenase and related enzymes in bovine epithelium. 5-Lipoxygenase was detected as an amplicon of 695 bp, which had 91% nucleotide sequence identity with human 5-Lipoxygenase and by Northern blot as a 3.0 kb mRNA. Leukotriene A4hydrolase was detected with the same techniques. The amino acid sequence of a 612 bp fragment was 90% identical with human leukotriene A4hydrolase and the size of the mRNA was 2.7 kb. The two enzymes were also detected in human corneal epithelium by reverse transcription-polymerase chain reaction.

  • cDNA cloning of 15-Lipoxygenase type 2 and 12-Lipoxygenases of bovine corneal epithelium
    Biochimica et Biophysica Acta, 1999
    Co-Authors: Maria Liminga, Ernst H. Oliw

    Abstract 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.

  • Arachidonate 15-Lipoxygenase in human corneal epithelium and 12- and 15-Lipoxygenases in bovine corneal epithelium: Comparison with other bovine 12-Lipoxygenase
    Biochimica et Biophysica Acta, 1994
    Co-Authors: Maria Liminga, Howard Sprecher, Lena Hörnsten, Ernst H. Oliw

    Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-Lipoxygenase and a 15-Lipoxygenase. The 12-Lipoxygenase was found in the microsomal fraction, while the 15-Lipoxygenase was mainly present in the cytosol (100 000 × g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosa-tetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid Lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 μ M. IC50 for the 12-Lipoxygenase was 0.3 μM. The bovine corneal 12-Lipoxygenase was compared with the 12-Lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized 14C-labelled linoleic acid and α-linolenic acid poorly (5–16%) in comparison with [l4C]arachidonic acid. [14C]Docosahexaenoic acid and [14C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyconal antibody against porcine leukocyte 12-Lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [14C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-Lipoxygenase and that bovine corneal epithelium contains both a 15-Lipoxygenase and a 12-Lipoxygenase. The corneal 12-Lipoxygenase appears to differ catalytically from earlier described bovine 12-Lipoxygenases.

Ambra Pozzi - One of the best experts on this subject based on the ideXlab platform.

  • Cyclooxygenases and Lipoxygenases in cancer
    Cancer and Metastasis Reviews, 2011
    Co-Authors: Claus Schneider, Ambra Pozzi

    Cancer initiation and progression are multistep events that require cell proliferation, migration, extravasation to the blood or lymphatic vessels, arrest to the metastatic site, and ultimately secondary growth. Tumor cell functions at both primary or secondary sites are controlled by many different factors, including growth factors and their receptors, chemokines, nuclear receptors, cell–cell interactions, cell–matrix interactions, as well as oxygenated metabolites of arachidonic acid. The observation that cyclooxygenases and Lipoxygenases and their arachidonic acid-derived eicosanoid products (prostanoids and HETEs) are expressed and produced by tumor cells, together with the finding that these enzymes can regulate cell growth, survival, migration, and invasion, has prompted investigators to analyze the roles of these enzymes in cancer progression. In this review, we focus on the contribution of cyclooxygenase- and Lipoxygenase-derived eicosanoids to tumor cell function in vitro and in vivo and discuss hope and tribulations of targeting these enzymes for cancer prevention and treatment.