Title of article :
Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies
Author/Authors :
Palmieri-Thiers، نويسنده , , Cynthia and Alberti، نويسنده , , Jean-Christophe and Canaan، نويسنده , , Stéphane and Brunini، نويسنده , , Virginie and Gambotti، نويسنده , , Claude and Tomi، نويسنده , , Félix and Oliw، نويسنده , , Ernst H. and Berti، نويسنده , , Liliane and Maury، نويسنده , , Jacques، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2011
Abstract :
Lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family.
Keywords :
site-directed mutagenesis , Homology-modeling , Olive lipoxygenase , Substrate-binding accessibility
Journal title :
Archives of Biochemistry and Biophysics
Journal title :
Archives of Biochemistry and Biophysics