title: Stereocontrol of arachidonic acid oxygenation by vertebrate lipoxygenases: newly cloned zebrafish lipoxygenase 1 does not follow the Ala-versus-Gly concept.
abstract: Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.
authors: Institute of Biochemistry, University Medicine Berlin-Charité, Monbijoustrasse 2, D-10117 Berlin, Germany.
authors: Christian
authors: C
authors: Jansen
authors: Katharina
authors: K
authors: Hofheinz
authors: Robert
authors: R
authors: Vogel
authors: Jana
authors: J
authors: Roffeis
authors: Monika
authors: M
authors: Anton
authors: Pallu
authors: P
authors: Reddanna
authors: Hartmut
authors: H
authors: Kuhn
authors: Matthias
authors: M
authors: Walther
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008084:Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D008253:Macaca mulatta; D051379:Mice; D057079:Pongo; D011817:Rabbits; D013045:Species Specificity; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D029961:Zebrafish Proteins; D016718:Arachidonic Acid; D008084:Lipoxygenase