eb3d466e5d24502562580e9bcf21e689 IntJouMol_a2023v24n12.pdf f71d4369cf7f0e018b058a336eb6368f23309b3a IntJouMol_a2023v24n12.pdf a7c3e978b6aae53c219494f7e334f990a16fe21fbadb3ed35adf85ed7c8e66d8 IntJouMol_a2023v24n12.pdf Title: Functional Characterization of Mouse and Human Arachidonic Acid Lipoxygenase 15B (ALOX15B) Orthologs and of Their Mutants Exhibiting Humanized and Murinized Reaction Specificities Subject: 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. Keywords: eicosanoids; lipids; metabolism; fatty acids; inflammation; atherosclerosis Author: Kumar R. Kakularam, Miquel Canyelles-Niño, Xin Chen, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jun 12 17:13:09 2023 CEST ModDate: Mon Jun 12 17:24:06 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 25 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5015050 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EPTQDJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 SBMXUO+VnURWPalladioL Type 1 Custom yes yes no 15 0 FIHTFO+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 DMFZHC+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 DKEXWI+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 PTVOAX+EURM10 Type 1 Builtin yes yes no 78 0 BCDEEE+PalatinoLinotype-Bold TrueType 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TrueType WinAnsi yes yes no 400 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 404 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 408 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 412 0 TimesNewRomanPSMT CID TrueType Identity-H yes no yes 419 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 434 0 XHAQEC+CMBSY10 Type 1 Builtin yes yes no 438 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-01 02:32:36 CET RepresentationInformation: IntJouMol_a2023v24n12.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-01-31 19:12:04 CET Size: 5015050 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 729 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Recombinant Expression of Mouse and Human ALOX15B Orthologs and of Selected Mutants Destination: subsection.2.1 Item: Title: Mouse and Human ALOX15B Variants Prefer C20 and C22 Polyenoic Fatty Acids over Linoleic Acid (C18) Destination: subsection.2.2 Item: Title: D602Y+V603H Exchange Partly Murinized the Reaction Products Formed by Human ALOX15B with EPA and AA but Altered the Product Pattern with DHA in a Different Way Destination: subsection.2.3 Item: Title: Y603D+H604V Mutation Humanized the Product Pattern of Mouse Alox15b with AA, EPA and DHA Destination: subsection.2.4 Item: Title: D602Y+V603H Exchange in Human ALOX15B and Y603D+H604V Mutation of Mouse Alox15b also Altered the Reaction Products of Linoleic Acid Oxygenation Destination: subsection.2.5 Item: Title: MD Simulations Suggest an Alternative Fatty Acid Binding at the Active Site of Human and Mouse ALOX15 Orthologs Destination: subsection.2.6 Item: Title: D602Y+V603H Exchange in Human ALOX15B and Y603D+H604V Mutation in the Mouse Ortholog also Altered the Substrate Behavior of HETE Isomer Destination: subsection.2.7 Item: Title: Wildtype Mouse Alox15b Forms Significant Amounts of 8,15-diHETE from AA during Long Term Incubations Destination: subsection.2.8 Item: Title: The Product Pattern of AA Oxygenation by Mouse Alox15b Depends on the Duration of the In Vitro Incubation Period and on the Amounts of Enzyme Added Destination: subsection.2.9 Item: Title: MD Simulations of 15-HETE and 8-HETE Binding Destination: subsection.2.10 Item: Title: Impact of Reaction Conditions on the Product Pattern of Mouse and Human ALOX15B Destination: subsection.2.11 Item: Title: Discussion Destination: section.3 Children: Item: Title: Degree of Novelty, Advancement of Knowledge, and Limitations Destination: subsection.3.1 Item: Title: Complex Ester Lipids as Substrate for Mouse and Human ALOX15B Destination: subsection.3.2 Item: Title: Impact of Reaction Conditions on the Product Pattern of ALOX15B Variants Destination: subsection.3.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Chemicals Destination: subsection.4.1 Item: Title: Cloning and Expression of Mouse and Human ALOXB Orthologs Destination: subsection.4.2 Item: Title: Mutagenesis Studies Destination: subsection.4.3 Item: Title: In Vitro Activity Assays and HPLC Analyses of the Reaction Products Destination: subsection.4.4 Item: Title: Miscellaneous Methods including Statistics Destination: subsection.4.5 Item: Title: Structural Modeling Destination: subsection.4.6 Item: Title: Docking Calculations Destination: subsection.4.7 Item: Title: MD Simulations Destination: subsection.4.8 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: Functional Characterization of Mouse and Human Arachidonic Acid Lipoxygenase 15B (ALOX15B) Orthologs and of Their Mutants Exhibiting Humanized and Murinized Reaction Specificities Author: Kumar R. Kakularam, Miquel Canyelles-Niño, Xin Chen, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn Subject: 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. 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