Functional characterization of novel ALOX15 orthologs representing key steps in mammalian evolution supports the Evolutionary Hypothesis of reaction specificity

Arachidonic acid lipoxygenases (ALOXs) are lipid-metabolizing enzymes that have been implicated in cell differentiation, but also in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Most mammalian genomes involve six or seven functional ALOX genes and among the corresponding ALOX-isoforms the ALOX15 orthologs are somewhat unique since they exhibit variable reaction specificity using arachidonic acid as substrate. The Evolutionary Hypothesis of mammalian ALOX15 reaction specificity (Prog. Lipid Res. 72, 55, 2018) suggests that ALOX15 orthologs of primates ranked higher in evolution than gibbons are 15-lipoxygenating enzymes. In contrast, mammals ranking lower than gibbons express dominantly 12-lipoxygenating lipoxygenases and gibbon ALOX15 constitutes a transition enzyme with pronounced dual reaction specificity. Here we predicted the reaction specificity of 95 different prototherian, metatherian and eutherian ALOX15 orthologs on the basis of their primary structures and characterized experimentally the reaction specificity of ten novel metatherian/eutherian enzymes representing different stages of mammalian evolution (gorilla, opossum, cape golden mole, dog, horseshoe bat, hedgehog, Sunda flying lemur, pika, chinchilla, kangaroo rat). We found that 97% of the currently sequenced mammalian ALOX15 including the enzymes of living and extinct hominids follow the Evolutionary Hypothesis. However, the ALOX15 orthologs of rabbits and of the Ord's kangaroo rat violate this mechanistic concept. Taken together, this data confirms the Evolutionary Hypothesis of ALOX15 reaction specificity and puts this concept on a more reliable experimental basis.