title: Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis.
abstract: Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.
authors.affiliation: University Medicine Berlin Charité, Monbijoustrasse 2, 10117 Berlin, Germany.
authors.forename: Sunitha
authors.initials: S
authors.lastname: Meruvu
authors.forename: Matthias
authors.initials: M
authors.lastname: Walther
authors.forename: Igor
authors.initials: I
authors.lastname: Ivanov
authors.forename: Sven
authors.initials: S
authors.lastname: Hammarström
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fürstenberger
authors.forename: Peter
authors.initials: P
authors.lastname: Krieg
authors.forename: Pallu
authors.initials: P
authors.lastname: Reddanna
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kuhn
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D014633:Valine
chemical_list: D006893:Hydroxyeicosatetraenoic Acids; D011994:Recombinant Proteins; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C027356:11-hydroxy-5,8,12,14-eicosatetraenoic acid; C047628:8-hydroxyeicosatetraenoic acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D001092:Arachidonate 12-Lipoxygenase; D014633:Valine