Alterations of lipoxygenase specificity by targeted substrate modificationand site-directed mutagenesis

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 alterat ions in the positional specificity can be reversed when bulky residues are introduced at the co end of the substrate. Simultaneous derivatisation of b oth ends of fatty acids forces a 15-LOX-catalysed 5-lipoxygenation and this reaction involves an inverse head-to-tail substrate orientation. In contra st, 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, con verts 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 subst rate-binding pocket. 15-LOX-catalysed 5-lipoxygenation involves an inverse substrate alignment but this may not be the case for 5-LOXs. Thus, both the ories for the mechanistic basis of 5-lipoxygenation (straight and inverse s ubstrate orientation) appear to be correct for different LOX isoforms. (C) 2001 Elsevier Science Ltd. All rights reserved.