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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.
Highlights
Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation
To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe390, Gly441, Ala455, and Val631 align with previously identified positional determinants of S-LOX isoforms
Mutagenesis of Positional Determinants Identified for Other LOX Isoforms—Previous mutagenesis studies on human and rabbit reticulocyte 15-LOXs identified Ile418 and Met419 and Phe353 and Ile593, respectively, as sequence determinants for the positional specificity [11, 12], but no information is currently available on the importance of these residues for the specificity of murine (12R)-LOX
Summary
Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. To investigate the structural basis for the stereochemical control mechanisms of murine (12R)-LOX more comprehensively, we applied a strategy that involves targeted substrate modification and site-directed mutagenesis For this purpose, we first identified putative sequence determinants for the reaction specificity by multiple amino acid sequence alignments and modified their side chains. Introduction of less bulky residues at Val631 induced both an increase in catalytic activities and specific alterations in product patterns, with (11R)-HETE methyl ester being the major oxygenation product These data indicate that Val631 constitutes a major sequence determinant for both the positional specificity and enantioselectivity of murine (12R)-LOX because the geometry of its side chain impacts the stereochemistry of both initial hydrogen abstraction and subsequent oxygen insertion
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