Functional Characterization of Mouse and Human Arachidonic Acid Lipoxygenase 15B (ALOX15B) Orthologs and of Their Mutants Exhibiting Humanized and Murinized Reaction Specificities
Kakularam, Kumar R. (Charité-Universitätsmedizin Berlin. Department of Biochemistry)
Canyelles-Niño, Miquel (Universitat Autònoma de Barcelona. Departament de Química)
Chen, Xin (Charité-Universitätsmedizin Berlin. Department of Biochemistry)
Lluch López, Josep Maria (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
González-Lafont, Àngels (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Kuhn, Hartmut (Charité-Universitätsmedizin Berlin. Department of Biochemistry)

Data:	2023
Resum:	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 variants. Wildtype human arachidonic acid lipoxygenase 15B converted arachidonic acid and eicosapentaenoic acid to their 15-hydroperoxy derivatives but the Asp602Tyr+Val603His exchange murinized the product pattern. The inverse mutagenesis strategy in mouse arachidonic acid lipoxygenase 15b (Tyr603Asp+His604Val exchange) humanized the product pattern with these substrates, but the situation was different with docosahexaenoic acid. Here, Tyr603Asp+His604Val substitution in mouse arachidonic acid lipoxygenase 15b also humanized the specificity but the inverse mutagenesis (Asp602Tyr+Val603His) did not murinize the human enzyme. With linoleic acid Tyr603Asp+His604Val substitution in mouse arachidonic acid lipoxygenase 15b humanized the product pattern but the inverse mutagenesis in human arachidonic acid lipoxygenase 15B induced racemic product formation. Amino acid exchanges at critical positions of human and mouse arachidonic acid lipoxygenase 15B orthologs humanized/murinized the product pattern with C20 fatty acids, but this was not the case with fatty acid substrates of different chain lengths. Asp602Tyr+Val603His exchange murinized the product pattern of human arachidonic acid lipoxygenase 15B with arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. An inverse mutagenesis strategy on mouse arachidonic acid lipoxygenase 15b (Tyr603Asp+His604Val exchange) did humanize the reaction products with arachidonic acid and eicosapentaenoic acid, but not with docosahexaenoic acid.
Ajuts:	Agencia Estatal de Investigación PID2020-113764GB-I00
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Atherosclerosis ; Eicosanoids ; Fatty acids ; Inflammation ; Lipids ; Metabolism
Publicat a:	International journal of molecular sciences, Vol. 24, Issue 12 (June 2023) , art. 10046, ISSN 1422-0067

DOI: 10.3390/ijms241210046
PMID: 37373195

25 p, 4.8 MB

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