Evolutionary alteration of ALOX15 specificity optimizes the biosynthesis of antiinflammatory and proresolving lipoxins

Abstract

ALOX15 (12/15-lipoxygenase) orthologs have been implicated in maturational degradation of intracellular organelles and in the biosynthesis of antiinflammatory and proresolving eicosanoids. Here we hypothesized that lower mammals (mice, rats, pigs) express 12-lipoxygenating ALOX15 orthologs. In contrast, 15-lipoxygenating isoforms are found in higher primates (orangutans, men), and these results suggest an evolution of ALOX15 specificity. To test this hypothesis we first cloned and characterized ALOX15 orthologs of selected Catarrhini representing different stages of late primate evolution and found that higher primates (men, chimpanzees) express 15-lipoxygenating orthologs. In contrast, lower primates (baboons, rhesus monkeys) express 12-lipoxygenating enzymes. Gibbons, which are flanked in evolution by rhesus monkeys (12-lipoxygenating ALOX15) and orangutans (15-lipoxygenating ALOX15), express an ALOX15 ortholog with pronounced dual specificity. To explore the driving force for this evolutionary alterations, we quantified the lipoxin synthase activity of 12-lipoxygenating (rhesus monkey, mouse, rat, pig, humIle418Ala) and 15-lipoxygenating (man, chimpanzee, orangutan, rabbit, ratLeu353Phe) ALOX15 variants and found that, when normalized to their arachidonic acid oxygenase activities, the lipoxin synthase activities of 15-lipoxygenating ALOX15 variants were more than fivefold higher (P < 0.01) [corrected]. Comparative molecular dynamics simulations and quantum mechanics/molecular mechanics calculations indicated that, for the 15-lipoxygenating rabbit ALOX15, the energy barrier for C13-hydrogen abstraction (15-lipoxygenation) was 17 kJ/mol lower than for arachidonic acid 12-lipoxygenation. In contrast, for the 12-lipoxygenating Ile418Ala mutant, the energy barrier for 15-lipoxygenation was 10 kJ/mol higher than for 12-lipoxygenation. Taken together, our data suggest an evolution of ALOX15 specificity, which is aimed at optimizing the biosynthetic capacity for antiinflammatory and proresolving lipoxins.

Keywords: eicosanoids; evolution; inflammation; lipoxygenase; protein design.

Fig. 1.

Triad determinants of mammalian ALOX15 orthologs including predicted and measured reaction specificity. The triad determinants of ALOX15 orthologs were retrieved from the databases. Only those entries were considered for further evaluation, for which the triad determinants have completely been sequenced. ALOX15 orthologs, for which the reaction specificity has been determined experimentally, are indicated in italic letters. On black background, those ALOX15 orthologs are indicated for which the reaction specificity has been determined for the first time in this study to our knowledge.

Fig. 2.

Simplified schematic view of late primate evolution. The main arachidonic acid oxygenation products are given in the gray shaded area. Unidentified product specificities are indicated by the question marks.

Fig. 3.

Partial sequence alignment of ALOX15 orthologs of selected mammals. The sequence regions of the triad determinants (BG1, SL, BG2) are given, and the critical amino acids are indicated in bold. The amino acid differences with potential impact on the reaction specificity are labeled on gray background.

Fig. 4.

Positional specificity of arachidonic acid oxygenation by ALOX15 orthologs representing late primate evolution. Arachidonate oxygenase activity assays and RP-HPLC analysis were carried out as described in Materials and Methods. Each chromatogram was scaled for the highest HETE peak. (A) Products formed by baboon ALOX15. (B) Products formed by chimpanzee ALOX15. (C) Products formed by gibbon ALOX15. (D) Products formed by human ALOX15.

Fig. 5.

Positional specificity of arachidonic acid oxygenation by gibbon ALOX15 mutants. Arachidonate oxygenase activity assay of gibbon ALOX15 and RP-HPLC analysis of the oxygenation products were carried out as described in Materials and Methods. Each chromatogram was scaled for the highest HETE peak.

Fig. 6.

Overlay (A) of WT ALOX15-AA (orange) and Ile418Ala ALOX15-AA (blue) active sites from two snapshots of the MD trajectories of both in silico models. The location of the arachidonic acid molecule with respect to the bottom of the cavity is shown in B and C. Arachidonic acid, the iron-bound hydroxyl ion (OH), and Fe are represented in sticks, and C₁₀, Ala-418, and Ile418 are represented in balls and sticks.

Scheme 1.

Evolutionary concept of ALOX15 development. ALOX15 orthologs of lower mammals are 12-lipoxygenating enzymes, whereas higher mammals including humans express 15-lipoxygenating orthologs. Hylobatidae (gibbons) appear to be the evolutionary switching point because they express an ALOX15 ortholog with pronounced dual reaction specificity.