Alterations of lipoxygenase specificity by targeted substrate modification and site-directed mutagenesis Original Research Article

Abstract Background: Mammalian lipoxygenases (LOXs) are categorised with respect to their positional specificity of arachidonic acid oxygenation. However, the mechanistic basis for this classification is not well understood. To gain a deeper insight into the structural basis of LOX specificity we determined the reaction characteristics of wild-type and mutant mammalian LOX isoforms with native and synthetic fatty acids substrates. Results: The rabbit 15-LOX is capable of catalysing major 12-lipoxygenation when the volume of the substrate-binding pocket is enlarged. These alterations in the positional specificity can be reversed when bulky residues are introduced at the ω end of the substrate. Simultaneous derivatisation of both ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contrast, for arachidonic acid 5-lipoxygenation by the human 5-LOX the substrate fatty acid may not be inversely aligned. The positional specificity of this isoenzyme may be related to its voluminous substrate-binding pocket. Site-directed mutagenesis, which leads to a reduction of active site volume, converts the 5-LOX to a 15-lipoxygenating enzyme species. Conclusions: The positional specificity of LOXs is not an invariant enzyme property but depends on the substrate structure and the volume of the substrate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both theories for the mechanistic basis of 5-lipoxygenation (straight and inverse substrate orientation) appear to be correct for different LOX isoforms.