A functional role identified for conserved charged residues at the active site entrance of lipoxygenase with double specificity

• Olive LOX1 active site entrance is surrounded by conserved positively charged residues.
• Substitution of R265, R268 and K283 by glutamine has an effect on kinetic parameters.
• These residues may interact with the substrate carboxylate.
• K283 could play a more important role attributable to its position on α2 helix.
• The role of these residues may be extended to all plant LOXs with double specificity.

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of free polyunsaturated fatty acids, producing 9-hydroperoxides or 13-hydroperoxides from linoleic and α-linolenic acids, and are called 9-LOX or 13-LOX, respectively. Some LOXs produce both 9- and 13-hydroperoxides. The models proposed to explain the reaction mechanism specificity fail to explain the “double specificity” character of these LOXs. In this study, we used the olive LOX1 with double specificity to investigate the implication of the charged residues R265, R268, and K283 in the orientation of the substrate into the active site. These residues are present in a conserved pattern around the entrance of the active site. Our results show that these residues are involved in the penetration of the substrate into the active site: this positive patch could capture the carboxylate end of the substrate, and then guide it into the active site. Due to its position on α2 helix, the residue K283 could have a more important role, its interaction with the substrate facilitating the motions of residues constituting the “cork of lipoxygenases” or the α2 helix, by disrupting putative hydrogen and ionic bonds.

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