Document Detail

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis.
MedLine Citation:
PMID:  16129665     Owner:  NLM     Status:  MEDLINE    
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.
Sunitha Meruvu; Matthias Walther; Igor Ivanov; Sven Hammarström; Gerhard Fürstenberger; Peter Krieg; Pallu Reddanna; Hartmut Kuhn
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't     Date:  2005-08-29
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  280     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2005 Nov 
Date Detail:
Created Date:  2005-10-31     Completed Date:  2005-12-28     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  36633-41     Citation Subset:  IM    
University Medicine Berlin Charité, Monbijoustrasse 2, 10117 Berlin, Germany.
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MeSH Terms
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / metabolism*
Amino Acid Sequence
Amino Acid Substitution
Arachidonate 12-Lipoxygenase / chemistry*,  genetics,  metabolism*
Binding Sites
Hydroxyeicosatetraenoic Acids / metabolism*
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed*
Recombinant Proteins / chemistry,  metabolism
Sequence Homology, Amino Acid
Substrate Specificity
Reg. No./Substance:
0/Hydroxyeicosatetraenoic Acids; 0/Recombinant Proteins; 59985-28-3/12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 7004-03-7/Valine; 73151-66-3/11-hydroxy-5,8,12,14-eicosatetraenoic acid; 73179-96-1/8-hydroxyeicosatetraenoic acid; 73945-47-8/15-hydroxy-5,8,11,13-eicosatetraenoic acid; EC 12-Lipoxygenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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