Eicosanoid biosynthesizing enzymes in Prototheria

Eicosanoids and related compounds are pleiotropic lipid mediators, which play a role in cell differentiation and in the pathogenesis of various diseases. The biosynthesis of these lipids has extensively been studied in highly developed mammals including humans but little is known about the formation of these mediators in more ancient Prototheria. We searched the genomes of two extant prototherian species (platypus, short-beaked echidna) for genes encoding for lipoxygenase- (ALOX) and prostaglandin synthase-isoforms (PTGS) and detected intact single copy genes for ALOX5, ALOX12, ALOX12B, ALOXE3, PTGS1 and PTGS2. Moreover, we identified two copies of ALOX15B genes (ALOX15B-1 and ALOX15B-2) but in echidna the ALOX15B-2 gene was structurally corrupted. Interestingly, in the two genomes ALOX15 genes were lacking. For functional characterization we expressed the prototherian ALOX15B isoforms and compared important enzyme characteristics of the wildtype proteins and of relevant enzyme mutants with those of human and mouse ALOX15B. Here we observed that the prototherian ALOX15B isoforms exhibit the same reaction specificity as their human ortholog. Mutagenesis of the Triad determinants did not alter the reaction specificity of the prototherian enzymes but modification of the Jisaka determinants murinized the catalytic properties. These data indicate that Prototheria exhibit an active eicosanoid metabolism. They express functional ALOX15B orthologs but lack ALOX15 genes. These observations and the previous findings that ALOX15 orthologs rarely occur in non-mammalian vertebrates such as fish and birds suggest that ALOX15 orthologs were introduced during Prototheria-Metatheria transition via an ALOX15B gene duplication and subsequent divergent enzyme evolution.