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IJMS | Free Full-Text | Functional Characterization of Mouse and Human Arachidonic Acid Lipoxygenase 15B (ALOX15B) Orthologs and of Their Mutants Exhibiting Humanized and Murinized Reaction Specificities

The arachidonic acid lipoxygenase 15B (ALOX15B) orthologs of men and mice form different reaction products when arachidonic acid is used as the substrate. Tyr603Asp+His604Val double mutation in mouse arachidonic acid lipoxygenase 15b humanized the product pattern and an inverse mutagenesis strategy murinized the specificity of the human enzyme. As the mechanistic basis for these functional differences, an inverse substrate binding at the active site of the enzymes has been suggested, but experimental proof for this hypothesis is still pending. Here we expressed wildtype mouse and human arachidonic acid lipoxygenase 15B orthologs as well as their humanized and murinized double mutants as recombinant proteins and analyzed the product patterns of these enzymes with different polyenoic fatty acids. In addition, in silico substrate docking studies and molecular dynamics simulation were performed to explore the mechanistic basis for the distinct reaction specificities of the different enzyme ....

Baden Wüberg , Bergisch Gladbach , Nordrhein Westfalen , United States , Miltenyi Biotec Gmb , Santa Clara , Carl Roth Gmb , Hewlett Packard , Daicel Corp , Graphpad Software , Agilent Technologies Inc , International Gmb , Mouse Alox , N Terminal Ile , Ester Lipids , Reaction Conditions , Product Pattern , Pfu Ultra , Agilent Technologies , Eurofins Genomics Germany Gmbh , Vitro Activity Assays , Carl Roth Gmbh , Miltenyi Biotec Gmbh , Serva Electrophoresis Gmbh , Image Analyzer , Intelligent Dark Box ,