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

  • 12 15 lipoxygenase oxidative modification of ldl and atherogenesis
    2001
    Co-Authors: Colin D. Funk, Tillmann Cyrus
    Abstract:

    Abstract Lipoxygenases comprise a family of non-heme iron-containing dioxygenases that stereospecifically insert molecular oxygen into free or esterified polyunsaturated fatty acids. The dual specificity 12/15-Lipoxygenases have been implicated in the oxidative modification of low-density lipoproteins and foam cell formation primarily based on in vitro studies. Recent in vivo data obtained with 12/15-lipoxygenase-deficient mice crossbred to apolipoprotein E-deficient mice have established a pro-atherogenic role for this pathway. In contrast, previous experiments with macrophage expressing 15-lipoxygenase transgenic rabbits have suggested an anti-atherogenic role. Possible explanations are presented that may elucidate these differences.

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

    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
    1999
    Co-Authors: Tillmann Cyrus, Rajendra K Tangirala, Sergio Fazio, Macrae F Linton, Joseph L Witztum, Daniel J. Rader, Colin D. Funk
    Abstract:

    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.

  • human 12 r lipoxygenase and the mouse ortholog molecular cloning expression and gene chromosomal assignment
    1998
    Co-Authors: Duxin Sun, Maeve Mcdonnell, Xinsheng Chen, Maha M Lakkis, Stuart N Isaacs, Sarah H Elsea, Pragna Patel, Colin D. Funk
    Abstract:

    Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an ≈80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian Lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situhybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.

  • cdna cloning expression mutagenesis of c terminal isoleucine genomic structure and chromosomal localizations of murine 12 Lipoxygenases
    1994
    Co-Authors: Xinsheng Chen, Usha Kurre, Nancy A Jenkins, Neal G Copeland, Colin D. Funk
    Abstract:

    Two types of 12-lipoxygenase that catalyze the transformation of arachidonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories. Here, we document, for the first time, a molecular characterization of both forms within the same species. The amino acid sequence of the murine platelet 12-lipoxygenase deduced from its cDNA is 58% identical to the murine spleen/leukocyte 12-lipoxygenase. Expression constructs carrying the cDNAs for the two 12-lipoxygenase forms were introduced into human embryonic kidney 293 cells. The platelet-type enzyme metabolized arachidonic acid exclusively to 12-HPETE, whereas the leukocyte-type enzyme formed both 12-HPETE and 15-hydro(pero)xyeicosatetraenoic acid in a ratio of approximately 3:1. Linoleic acid was metabolized to a similar extent by the latter enzyme to 13-hydro(pero)xyoctadecadienoic acid but not by the platelet enzyme. Mutagenesis and deletion of the highly conserved lipoxygenase C-terminal isoleucine (Ile663), a residue believed to be involved in the non-heme iron atom coordination of all Lipoxygenases, was performed. Deletion of Ile663 and substitution with most amino acids abolished enzyme activity. Only a valine substitution retained significant activity. These findings would tend to indicate a stringent requirement for the proper spatial alignment and folding of the C-terminal chain back into the core of the enzyme to interact with the iron atom by analogy with the recently determined crystal structure of a soybean lipoxygenase (Boyington, J. C., Gaffney, B. J., and Amzel, L. M. (1993) Science 260, 1482-1486). The platelet-type and leukocyte-type 12-lipoxygenase genes were cloned from a murine 129 Sv genomic library. Both genes are divided into a similar 14-exon/13-intron format, with the platelet-type gene being approximately twice the size of the leukocyte-type gene (13 versus 7.5 kilobases). A segment of a third gene was also isolated and probably represents a pseudogene derivative of either of these 12-lipoxygenase genes. All three genes were mapped to the central region of mouse chromosome 11 in a region of homology with human chromosome 17. Antibodies prepared against the two forms of 12-lipoxygenase revealed the differential distribution of the two enzymes throughout the mouse.

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

  • mammalian Lipoxygenases and their biological relevance
    2015
    Co-Authors: Hartmut Kühn, Swathi Banthiya, Klaus Van Leyen
    Abstract:

    Abstract Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated not only in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOXs oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, Lipoxygenases are key players in the regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian Lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. This article is part of a Special Issue entitled “Oxygenated metabolism of PUFA: analysis and biological relevance”.

  • evolutionary aspects of Lipoxygenases and genetic diversity of human leukotriene signaling
    2015
    Co-Authors: Thomas Horn, Hartmut Kühn, Susan Adel, Ralf R Schumann, Saubashya Sur, Kumar Reddy Kakularam, Aparoy Polamarasetty, Pallu Redanna, Dagmar Heydeck
    Abstract:

    Leukotrienes are pro-inflammatory lipid mediators, which are biosynthesized via the lipoxygenase pathway of the arachidonic acid cascade. Lipoxygenases form a family of lipid peroxidizing enzymes and human lipoxygenase isoforms have been implicated in the pathogenesis of inflammatory, hyperproliferative (cancer) and neurodegenerative diseases. Lipoxygenases are not restricted to humans but also occur in a large number of pro- and eucaryotic organisms. Lipoxygenase-like sequences have been identified in the three domains of life (bacteria, archaea, eucarya) but because of lacking functional data the occurrence of catalytically active Lipoxygenases in archaea still remains an open question. Although the physiological and/or pathophysiological functions of various lipoxygenase isoforms have been studied throughout the last three decades there is no unifying concept for the biological importance of these enzymes. In this review we are summarizing the current knowledge on the distribution of Lipoxygenases in living single and multicellular organisms with particular emphasis to higher vertebrates and will also focus on the genetic diversity of enzymes and receptors involved in human leukotriene signaling.

  • functional characterization of genetic enzyme variations in human Lipoxygenases
    2013
    Co-Authors: Thomas Horn, Monika Anton, Kumar Reddy Kakularam, P Reddanna, Constanze Richter, Hartmut Kühn
    Abstract:

    Mammalian Lipoxygenases play a role in normal cell development and differentiation but they have also been implicated in the pathogenesis of cardiovascular, hyperproliferative and neurodegenerative diseases. As lipid peroxidizing enzymes they are involved in the regulation of cellular redox homeostasis since they produce lipid hydroperoxides, which serve as an efficient source for free radicals. There are various epidemiological correlation studies relating naturally occurring variations in the six human lipoxygenase genes (SNPs or rare mutations) to the frequency for various diseases in these individuals, but for most of the described variations no functional data are available. Employing a combined bioinformatical and enzymological strategy, which included structural modeling and experimental site-directed mutagenesis, we systematically explored the structural and functional consequences of non-synonymous genetic variations in four different human lipoxygenase genes (ALOX5, ALOX12, ALOX15, and ALOX15B) that have been identified in the human 1000 genome project. Due to a lack of a functional expression system we resigned to analyze the functionality of genetic variations in the hALOX12B and hALOXE3 gene. We found that most of the frequent non-synonymous coding SNPs are located at the enzyme surface and hardly alter the enzyme functionality. In contrast, genetic variations which affect functional important amino acid residues or lead to truncated enzyme variations (nonsense mutations) are usually rare with a global allele frequency<0.1%. This data suggest that there appears to be an evolutionary pressure on the coding regions of the lipoxygenase genes preventing the accumulation of loss-of-function variations in the human population.

  • the n terminal β barrel domain of mammalian Lipoxygenases including mouse 5 lipoxygenase is not essential for catalytic activity and membrane binding but exhibits regulatory functions
    2011
    Co-Authors: Matthias Walther, Katharina Hofheinz, Robert Vogel, Jana Roffeis, Hartmut Kühn
    Abstract:

    Mammalian Lipoxygenases (LOXs) have been implicated in cell differentiation and in the pathogenesis of inflammatory and hyperproliferative diseases. The available structural information indicated that Lipoxygenases constitute single polypeptide chain enzymes consisting of a small N-terminal β-barrel domain and a larger C-terminal subunit that harbors the catalytic non-heme iron. Because of its structural similarity to C2-domains of lipases the N-terminal β-barrel domain of Lipoxygenases, which comprises about 110 amino acids, has been implicated in membrane binding and activity regulation. To explore the functional relevance of the C2-domain in more detail and to develop a more comprehensive hypothesis on the biological role of this structural subunit we performed gene technical truncation on various mammalian LOX isoforms (12/15-LOXs of various species, human 15-LOX2, mouse 5-LOX) and quantified catalytic activity and membrane binding properties of the truncated recombinant enzyme species. We found that the C2-domain is not essential for catalytic activity and does hardly impact reaction specificity. Truncated enzyme species exhibit impaired membrane binding properties and altered reaction kinetics. Taken together, our data suggests a regulatory importance of the N-terminal β-barrel domain for mammalian lipoxygenase isoforms.

  • structural biology of mammalian Lipoxygenases enzymatic consequences of targeted alterations of the protein structure
    2005
    Co-Authors: Hartmut Kühn, Jan Saam, Sebastian Eibach, Hermanngeorg Holzhutter, Igor Ivanov, Matthias Walther
    Abstract:

    Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the pathogenesis of diseases with major health political relevance (bronchial asthma, atherosclerosis, cancer, and osteoporosis). The crystal structures of one mammalian lipoxygenase and of two plant isoenzymes have been solved and the structural bases of important enzyme properties (reaction specificity, membrane binding, and suicidal inactivation) have been investigated in the past. This review will briefly summarize our current understanding on the structural biology of the most important mammalian lipoxygenase isoforms and will also address selected mechanistic features of the lipoxygenase reaction.

Ivo Feussner - One of the best experts on this subject based on the ideXlab platform.

  • lipoxygenase 2 from cyanothece sp controls dioxygen insertion by steric shielding and substrate fixation
    2017
    Co-Authors: Julia Newie, Piotr Neumann, Martin Werner, Ricardo A Mata, Ralf Ficner, Ivo Feussner
    Abstract:

    The biological function of Lipoxygenases depends on the regio and stereo specific formation of fatty acid-derived hydroperoxides and different concepts exist to explain the mechanism that directs dioxygen to a specific carbon atom within the substrate. Here, we report the 1.8 A resolution crystal structure of a cyanobacterial lipoxygenase that produces bis-allylic hydroperoxides (CspLOX2). Site directed mutagenesis experiments combined with computational approaches reveal that residues around the active site direct dioxygen to a preferred carbon atom and stereo configuration in the substrate fatty acid. Modulating the cavity volume around the pentadiene system of linoleic acid shifted the product formation towards 9S-, 9R-, 13S- or 13R-hydroperoxides in correlation with the site of mutation, thus decreasing the amount of the bis-allylic 11R-hydroperoxide. Decreasing the channel size of a 9R-lipoxygenase (CspLOX1) on the other hand could in turn induce formation of the bis-allylic 11R-hydroperoxide. Together this study suggests that an active site clamp fixing the pentadiene system of the substrate together with steric shielding controls the stereo and regio specific positioning of dioxygen at all positions of the reacting pentadiene system of substrate fatty acids.

  • a bisallylic mini lipoxygenase from cyanobacterium cyanothece sp that has an iron as cofactor
    2010
    Co-Authors: Alexandra Z Andreou, Cornelia Göbel, Mats Hamberg, Ivo Feussner
    Abstract:

    Lipoxygenases are enzymes that are found ubiquitously in higher animals and plants, but have only recently been identified in a number of bacteria. The genome of the diazotrophic unicellular cyanobacterium Cyanothece sp. harbors two genes with homology to Lipoxygenases. Here we describe the isolation of one gene, formerly named csplox2. It was cloned, and the protein was expressed in Escherichia coli and purified. The purified enzyme belongs to the group of prokaryotic mini Lipoxygenases, because it had a molecular mass of 65 kDa. Interestingly, it catalyzed the conversion of linoleic acid, the only endogenously found polyunsaturated fatty acid, primarily to the bisallylic hydroperoxide 11R-hydroperoxyoctadecadienoic acid. This product had previously only been described for the manganese lipoxygenase from the take all fungus, Gaeumannomyces graminis. By contrast, CspLOX2 was shown to be an iron lipoxygenase. In addition, CspLOX2 formed a mixture of typical conjugated lipoxygenase products, e.g. 9R- and 13S-hydroperoxide. The conversion of linoleic acid took place with a maximum reaction rate of 31 s−1. Incubation of the enzyme with [(11S)-2H]linoleic acid led to the formation of hydroperoxides that had lost the deuterium label, thus suggesting that CspLOX2 catalyzes antarafacial oxygenation as opposed to the mechanism of manganese lipoxygenase. CspLOX2 could also oxidize diarachidonylglycerophosphatidylcholine with similar specificity as the free fatty acid, indicating that binding of the substrate takes place with a “tail-first” orientation. We conclude that CspLOX2 is a novel iron mini-lipoxygenase that catalyzes the formation of bisallylic hydroperoxide as the major product.

  • physcomitrella patens has Lipoxygenases for both eicosanoid and octadecanoid pathways
    2009
    Co-Authors: Aldwin M Anterola, Ivo Feussner, Cornelia Göbel, Ellen Hornung, George E Sellhorn, Howard D Grimes
    Abstract:

    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.

  • A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens.
    2004
    Co-Authors: Toralf Senger, Thomas Wichard, Susan Kunze, Cornelia Göbel, Jens Lerchl, Georg Pohnert, Ivo Feussner
    Abstract:

    Abstract A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to Lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 Lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C18-C22 fatty acids giving rise to even more LOX-derived products. In contrast to other Lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

  • lipid peroxidation during the hypersensitive response in potato in the absence of 9 Lipoxygenases
    2003
    Co-Authors: Cornelia Göbel, Ivo Feussner, Sabine Rosahl
    Abstract:

    Hypersensitive cell death is an important defense reaction of plants to pathogen infection and is accompanied by lipid peroxidation processes. These may occur non-enzymatically by the action of reactive oxygen species or may be catalyzed by enzymes such as alpha-dioxygenases, Lipoxygenases, or peroxidases. Correlative data showing increases in 9-lipoxygenase products in hyper-sensitively reacting cells have so far suggested that a large part of lipid peroxidation is mediated by a specific set of 9-Lipoxygenases. To address the significance of 9-Lipoxygenases for this type of pathogen response in potato, RNA interference constructs of a specific pathogen-induced potato 9-lipoxygenase were transferred to potato plants. Significantly reduced 9-lipoxygenase transcript levels were observed in transgenic plants after pathogen treatment. In addition, 9-lipoxygenase activity was hardly detectable, and levels of 9-lipoxygenase-derived oxylipins were reduced up to 12-fold after pathogen infection. In contrast to wild type plants, high levels of non-enzymatically as well as 13-lipoxygenase-derived oxylipins were present in 9-lipoxygenase-deficient plants. From this we conclude that during the normal hypersensitive response in potato, lipid peroxidation may occur as a controlled and directed process that is facilitated by the action of a specific 9-lipoxygenase. If 9-lipoxygenase-mediated formation of hydroperoxides is repressed, autoxidative lipid peroxidation processes and 13-lipoxygenase-mediated oxylipins synthesis become prominent. The unaltered timing and extent of necrosis formation suggests that the origin of lipid hydroperoxides does not influence pathogen-induced cell death in potato.

Garreta Gambús I Albert - One of the best experts on this subject based on the ideXlab platform.

  • Cristal·lització de la lipoxigenasa de "Pseudomonas aeruginosa" 42A2 i estudi filogenètic de les subfamílies de les lipoxigenases (Tesi)
    2010
    Co-Authors: Garreta Gambús I Albert
    Abstract:

    [cat] Les lipoxigenases són conegudes com les responsables de la catàlisi d'oxigenació molecular d’àcids grassos poli-insaturats que forma un producte hydroperoxid (tòxic) que es pot convertir en una gran varietat de metabòlits secundaris. Durant anys, la presència de lipoxgenases ha estat descrita únicament en organismes eucariotes, mamífers, plantes, petits invertebrats marins i fongs. La funció biològica de les lipoxigenases eucariotes ha estat àmpliament estudiada. Estan involucrades en la síntesi de molècules de senyalització com els leucotriens, en mamífers, o en la de l’àcid jasmònic, en plantes. L’any 1964, Shimahara, publicava la presència d’una lipoxigenasa en un bacteri gram-negatiu aïllat de les escombraries. Encara avui són poques les informacions referents a lipoxigenases bacterianes que s'han publicat, alguns exemples són les de Thermoactinomyces vulgaris, Pseudomonas i Nostoc punctiforme. Els anàlisis que s’han realitzat utilitzant el programa BLAST, indiquen que hi ha gens “LOX-like” en Shewanella, Burkolderia, Nitrosomonas i Myxococcus, però la funció biològica de lipoxigenases procariotes encara no s'ha aclarit. No obstant, les lipoxigenases publicades en la base de dades “gene data-bank” són d’organismes ambientals de medi marí i de sòls, això suggereix que les lipoxigenases poden tenir un paper important en la biodegradació. En aquest estudi es reporta la primera estructura molecular d'una lipoxigenasa procariota i un estudi preliminar de la filogènia d’aquest grup de proteïnes.[eng] The Lipoxygenases are known as responsible for the reaction of oxygenation of poly-unsaturated fatty acid producing hydroperoxid acids (toxic) that can be converted into a different secondary metabolites. For years, the presence of Lipoxygenases has been described only in eukaryotes, mammals, plants, fungi and small invertebrates. The biological function of eukaryotic Lipoxygenases has been widely studied. They are involved in the synthesis of leukotrienes as signaling molecules in mammals, or in the jasmonic acid in plants. In 1964, Shimahara, published the existence of Lipoxygenases of gram-negative bacteria isolated from the trash. Even today there are few data concerning bacterial Lipoxygenases published. Some examples are those of Thermoactinomyces vulgaris, Pseudomonas and Nostoc punctiforme. The analysis were performed using the BLAST program and the results indicate that there are "LOX-like" genes in Shewanella, Burkolderia, Nitrosomonas and Myxococcus, but the biological function of prokaryotic Lipoxygenases is not yet clear. However, Lipoxygenases published in the database "gene data-bank" are marine organisms and environmental grounds, suggesting that the Lipoxygenases can play an important role in biodegradation. This study reports the first molecular structure of a prokaryotic lipoxygenasa and a preliminary study of the phylogeny of this group of proteins

  • Cristal•lització de la lipoxigenasa de "Pseudomonas aeruginosa" 42A2 i estudi filogenètic de les subfamílies de les lipoxigenases
    2010
    Co-Authors: Garreta Gambús I Albert
    Abstract:

    Les lipoxigenases són conegudes com les responsables de la catàlisi d'oxigenació molecular d’àcids grassos poli-insaturats que forma un producte hydroperoxid (tòxic) que es pot convertir en una gran varietat de metabòlits secundaris. Durant anys, la presència de lipoxgenases ha estat descrita únicament en organismes eucariotes, mamífers, plantes, petits invertebrats marins i fongs. La funció biològica de les lipoxigenases eucariotes ha estat àmpliament estudiada. Estan involucrades en la síntesi de molècules de senyalització com els leucotriens, en mamífers, o en la de l’àcid jasmònic, en plantes. L’any 1964, Shimahara, publicava la presència d’una lipoxigenasa en un bacteri gram-negatiu aïllat de les escombraries. Encara avui són poques les informacions referents a lipoxigenases bacterianes que s'han publicat, alguns exemples són les de Thermoactinomyces vulgaris, Pseudomonas i Nostoc punctiforme. Els anàlisis que s’han realitzat utilitzant el programa BLAST, indiquen que hi ha gens “LOX-like” en Shewanella, Burkolderia, Nitrosomonas i Myxococcus, però la funció biològica de lipoxigenases procariotes encara no s'ha aclarit. No obstant, les lipoxigenases publicades en la base de dades “gene data-bank” són d’organismes ambientals de medi marí i de sòls, això suggereix que les lipoxigenases poden tenir un paper important en la biodegradació. En aquest estudi es reporta la primera estructura molecular d'una lipoxigenasa procariota i un estudi preliminar de la filogènia d’aquest grup de proteïnes.The Lipoxygenases are known as responsible for the reaction of oxygenation of poly-unsaturated fatty acid producing hydroperoxid acids (toxic) that can be converted into a different secondary metabolites. For years, the presence of Lipoxygenases has been described only in eukaryotes, mammals, plants, fungi and small invertebrates. The biological function of eukaryotic Lipoxygenases has been widely studied. They are involved in the synthesis of leukotrienes as signaling molecules in mammals, or in the jasmonic acid in plants. In 1964, Shimahara, published the existence of Lipoxygenases of gram-negative bacteria isolated from the trash. Even today there are few data concerning bacterial Lipoxygenases published. Some examples are those of Thermoactinomyces vulgaris, Pseudomonas and Nostoc punctiforme. The analysis were performed using the BLAST program and the results indicate that there are "LOX-like" genes in Shewanella, Burkolderia, Nitrosomonas and Myxococcus, but the biological function of prokaryotic Lipoxygenases is not yet clear. However, Lipoxygenases published in the database "gene data-bank" are marine organisms and environmental grounds, suggesting that the Lipoxygenases can play an important role in biodegradation. This study reports the first molecular structure of a prokaryotic lipoxygenasa and a preliminary study of the phylogeny of this group of proteins

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

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

    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.

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

    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.