40 research outputs found
Mutations of triad determinants changes the substrate alignment at the catalytic center of human ALOX5.
No full textFor the specificity of ALOX15 orthologs of different mammals the geometry of the amino acids Phe353, Ile418, Met419 and Ile593 (?triad determinants?) is important and mutagenesis of these residues altered the reaction specificity of these enzymes. Here we expressed wildtype human ALOX5 and its F359W/A424I/N425M/A603I mutant in Sf9 insect cells and characterized the catalytic differences of the two enzyme variants. We found that wildtype ALOX5 converted arachidonic acid mainly to 5(S)-HpETE. In contrast, 15(S)- and 8(S)-H(p)ETE were formed by the mutant enzyme. In addition to arachidonic acid, wildtype ALOX5 accepted EPA as substrate but C18 fatty acids were not oxygenated. The quadruple mutant also accepted linoleic acid, alpha- and gamma-linolenic acid as substrate. Structural analysis of the oxygenation products and kinetic studies with stereospecifically labeled 11(S)- and 11(R)-deutero linoleic acid suggested alternative ways of substrate orientation at the active site. In silico docking studies, molecular dynamics simulations and QM/MM calculations confirmed this hypothesis. These data indicate that ?triad determinant? mutagenesis alters the catalytic properties of ALOX5 abolishing its leukotriene synthase activity but improving its biosynthetic capacity for pro-resolving lipoxins.Fil: Ivanov, Igor. Russian Technological University;Fil: Golovanov, Alexey B.. Russian Technological University;Fil: Ferretti, Cristián Alejandro. Universidad Nacional del Litoral. Instituto de Química Aplicada del Litoral. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Química Aplicada del Litoral.; ArgentinaFil: Canyelles-Niño, Miquel. Universitat Autònoma de Barcelona; EspañaFil: Heydeck, Dagmar. Humboldt University Berlin;Fil: Stehling, Sabine. Humboldt University Berlin;Fil: Lluch, José M.. Universitat Autònoma de Barcelona; EspañaFil: González Lafont, Ángels. Universitat Autònoma de Barcelona; EspañaFil: Kühn, Hartmut. Humboldt University Berlin
Functional Characterization of Novel Bony Fish Lipoxygenase Isoforms and Their Possible Involvement in Inflammation
No full textEicosanoids and related compounds are pleiotropic lipid mediators, which are biosynthesized in mammals via three distinct metabolic pathways (cyclooxygenase pathway, lipoxygenase pathway, epoxygenase pathway). These mediators have been implicated in the pathogenesis of inflammatory diseases and drugs interfering with eicosanoid signaling are currently available as antiphlogistics. Eicosanoid biosynthesis has well been explored in mammals including men, but much less detailed information is currently available on eicosanoid biosynthesis in other vertebrates including bony fish. There are a few reports in the literature describing the expression of arachidonic acid lipoxygenases (ALOX isoforms) in several bony fish species but except for two zebrafish ALOX-isoforms (zfALOX1 and zfALOX2) bony fish eicosanoid biosynthesizing enzymes have not been characterized. To fill this gap and to explore the possible roles of ALOX15 orthologs in bony fish inflammation we cloned and expressed putative ALOX15 orthologs from three different bony fish species (N. furzeri, P. nyererei, S. formosus) as recombinant N-terminal his-tag fusion proteins and characterized the corresponding enzymes with respect to their catalytic properties (temperature-dependence, activation energy, pH-dependence, substrate affinity and substrate specificity with different polyenoic fatty acids). Furthermore, we identified the chemical structure of the dominant oxygenation products formed by the recombinant enzymes from different free fatty acids and from more complex lipid substrates. Taken together, our data indicate that functional ALOX isoforms occur in bony fish but that their catalytic properties are different from those of mammalian enzymes. The possible roles of these ALOX-isoforms in bony fish inflammation are discussed
Functional characterization of a novel arachidonic acid 12S-lipoxygenase in the halotolerant bacterium Myxococcus fulvus exhibiting complex social living patterns
No full textLipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and mammals. These enzymes are also expressed in lower multicellular organisms but here they are not widely distributed. In bacteria, lipoxygenases rarely occur and evaluation of the currently available bacterial genomes suggested that <0.5% of all sequenced bacterial species carry putative lipoxygenase genes. We recently rescreened the public bacterial genome databases for lipoxygenase‐like sequences and identified two novel lipoxygenase isoforms (MF‐LOX1 and MF‐LOX2) in the halotolerant Myxococcus fulvus. Both enzymes share a low degree of amino acid conservation with well‐characterized eukaryotic lipoxygenase isoforms but they involve the catalytically essential iron cluster. Here, we cloned the MF‐LOX1 cDNA, expressed the corresponding enzyme as N‐terminal hexa‐his‐tag fusion protein, purified the recombinant enzyme to electrophoretic homogeneity, and characterized it with respect to its protein‐chemical and enzymatic properties. We found that M. fulvus expresses a catalytically active intracellular lipoxygenase that converts arachidonic acid and other polyunsaturated fatty acids enantioselectively to the corresponding n‐9 hydroperoxy derivatives. The enzyme prefers C20‐ and C22‐polyenoic fatty acids but does not exhibit significant membrane oxygenase activity. The possible biological relevance of MF‐LOX1 will be discussed in the context of the suggested concepts of other bacterial lipoxygenases
Secreted lipoxygenase from Pseudomonas aeruginosa exhibits biomembrane oxygenase activity and induces hemolysis in human red blood cells
No full textRegulation of cellular 15‐lipoxygenase activity on pretranslational, translational, and posttranslational levels
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