Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes |
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Authors: | Susan Adel,Katharina Hofheinz,Dagmar Heydeck,Hartmut Kuhn,Ann-Kathrin Hä fner |
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Affiliation: | 1. Institute of Biochemistry, Charite — University Medicine Berlin — Charité, CCO-Building, Virchowweg 6, 10117 Berlin, Germany;2. Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany |
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Abstract: | 5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (> 95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity. |
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Keywords: | LOX, lipoxygenase humALOX5, human 5-lipoxygenase mouAlox5, mouse 5-lipoxygenase zfhAlox5, zebrafish 5-lipoxygenase 5-HETE, 5-hydroxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid 8-HETE, 8-hydroxy-5,9,11,14(Z,E,Z,Z)-eicosatetraenoic acid 12-HETE, 12-hydroxy-5,8,10,14(Z,Z,E,Z)-eicosatetraenoic acid 15-HETE, 15-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid RP-HPLC, reverse phase high performance liquid chromatography IPTG, isopropyl-β-D-1-thiogalactopyranoside |
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