© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim The reaction specificity of lipoxygenases is of physiological relevance since the various oxygenation products exhibit different biological activities. Among mammalian ALOX15 orthologs there are arachidonic acid 12- and 15-lipoxygenating enzymes and recent studies suggested an evolutionary switch in that reaction specificity during late primate development. Previous reports showed that 12-lipoxygenating ALOX15 orthologs can be converted to 15-lipoxygenating enzymes by site-directed mutagenesis of some sequence determinants. Unfortunately, the molecular basis for those alterations are not well understood. Here, the arachidonic acid 12-lipoxygenating N-terminal truncation variant of pig ALOX15, for which a crystal structure is available, was used to explore the catalytic mechanism of the specificity switch induced by mutagenesis of Val418 and Val419 sequence determinants. We found that Val418Ile+Val419Met double mutant is dominantly 15-lipoxygenating. Docking and MD simulations, and quantum mechanics/molecular mechanics calculations indicated that the wildtype energy barrier for arachidonic acid 15-lipoxygenation is 3.4 kcal mol−1 higher than for 12-lipoxygenation. In contrast, for the Val418Ile+Val419Met double mutant the energy barrier for 12-lipoxygenation is 6.0 kcal mol−1 higher than for 15-lipoxygenation. Our data suggest that enzyme–substrate complex geometries determine the value of these energy barriers and, as a consequence, the reaction specificity of ALOX15 orthologs.
|Journal||Chemistry - A European Journal|
|Publication status||Published - 19 Jan 2018|
- biological chemistry