Mutagenesis of triad determinants of rat Alox15 alters the specificity of fatty acid and phospholipid oxygenation.
Among lipoxygenases ALOX15 orthologs are somewhat peculiar because of their capability of oxygenating polyenoic fatty acids even if they are incorporated in complex lipid-protein assemblies. ALOX15 orthologs of different species have been characterized before, but little is known about the corresponding rat enzyme. Since rats are frequently employed as models in biomedical research we expressed rat Alox15 as recombinant protein in pro- and eukaryotic expression systems and characterized the enzyme with respect to its enzymatic properties. The enzyme oxygenated free arachidonic acid mainly to 12S-HpETE with 15S-HpETE only contributing 10% to the product mixture. Multiple directed mutagenesis studies indicated applicability of the triad concept with particular importance of Leu353 and Ile593 as specificity determinants. Ala404Gly exchange induced subtle alterations in enantioselectivity suggesting partial applicability of the Coffa/Brash concept. Wildtype rat Alox15 and its 15-lipoxygenating Leu353Phe mutant are capable of oxygenating ester lipids of biomembranes and high-density lipoproteins. For the wildtype enzyme 13S-HODE and 12S-HETE were identified as major oxygenation products but for the Leu353Phe mutant 13S-HODE and 15S-HETE prevailed. These data indicate for the first time that mutagenesis of triad determinants modifies the reaction specificity of ALOX15 orthologs with free fatty acids and complex ester lipids in a similar way.
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany; Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, 832 32 Bratislava, Slovakia.
Mária
M
Pekárová
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany.
Hartmut
H
Kuhn
Department of Cell and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University, Kalinčiakova 8, 832 32 Bratislava, Slovakia.
Lýdia
L
Bezáková
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany.
Christoph
C
Ufer
Institute of Biochemistry, University Medicine Berlin - Charité, Chariteplatz 1, D-10117 Berlin, Germany. Electronic address: dagmar.heydeck@charite.de.
Dagmar
D
Heydeck
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D045744:Cell Line, Tumor; D005227:Fatty Acids / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D051336:Mitochondrial Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity
D005227:Fatty Acids; D007527:Isoenzymes; D010743:Phospholipids; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase
ALOX15; Biomembranes; Eicosanoids; Lipoproteins; Oxidative stress; Reaction mechanism