title: The rat leukocyte-type 12-lipoxygenase exhibits an intrinsic hepoxilin A3 synthase activity.
abstract: Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A(3) synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A(3). 12S-lipoxygenase and hepoxilin A(3) synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A(3) synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A(3) synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A(3) synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A(3) synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.
authors.affiliation: Eicosanoid and Lipid Research Division and Centre of Experimental Gynecology and Breast Research, University Medical Centre Berlin, Campus Benjamin Franklin, Free University of Berlin, D-12200 Berlin, Germany. nigam@zedat.fu-berlin.de
authors.forename: Santosh
authors.initials: S
authors.lastname: Nigam
authors.forename: Shankarnarayanan
authors.initials: S
authors.lastname: Patabhiraman
authors.forename: Roberto
authors.initials: R
authors.lastname: Ciccoli
authors.forename: Ganchimeg
authors.initials: G
authors.lastname: Ishdorj
authors.forename: Kristin
authors.initials: K
authors.lastname: Schwarz
authors.forename: Branka
authors.initials: B
authors.lastname: Petrucev
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kühn
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment
chemical_list: D001095:Arachidonic Acids; D007527:Isoenzymes; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase; D019746:Intramolecular Oxidoreductases; C473385:hepoxilin synthase