Eicosanoid biosynthesis in marine mammals

After 300 million years of evolution, the first land‐living mammals reentered the marine environment some 50 million years ago. The driving forces for this dramatic lifestyle change are still a matter of discussion but the struggle for food resources and the opportunity to escape predators probably contributed. Reentering the oceans requires metabolic adaption putting evolutionary pressure on a number of genes. To explore whether eicosanoid signaling has been part of this adaptive response, we first explored whether the genomes of marine mammals involve functional genes encoding for key enzymes of eicosanoid biosynthesis. Cyclooxygenase (COX) and lipoxygenase (ALOX) genes are present in the genome of all marine mammals tested. Interestingly, ALOX12B, which has been implicated in skin development of land‐living mammals, is lacking in whales and dolphins and genes encoding for its sister enzyme (ALOXE3) involve premature stop codons and/or frameshifting point mutations, which interrupt the open reading frames. ALOX15 orthologs have been detected in all marine mammals, and the recombinant enzymes exhibit similar catalytic properties as those of land‐living species. All marine mammals express arachidonic acid 12‐lipoxygenating ALOX15 orthologs, and these data are consistent with the Evolutionary Hypothesis of ALOX15 specificity. These enzymes exhibit membrane oxygenase activity and introduction of big amino acids at the triad positions altered the reaction specificity in favor of arachidonic acid 15‐lipoxygenation. Thus, the ALOX15 orthologs of marine mammals follow the Triad concept explaining their catalytic specificity.

Eicosanoids and related compounds are lipid-signaling molecules, which have been implicated in cell physiology and in the pathogenesis of hyperproliferative, inflammatory, and neurodegenerative diseases [12][13][14]. They are biosynthesized from arachidonic acid and other polyenoic fatty acids (PUFAs) via three different pathways: (i) cyclooxygenase (COX) pathway leading to the formation of pro-inflammatory prostaglandins [15], (ii) lipoxygenase (ALOX) pathway leading to the formation of pro-inflammatory leukotrienes, but also to anti-inflammatory mediators such as resolvins and maresins [16], and (iii) cytochrome P450 (Cyt450) pathway leading to the formation of epoxyeicosanoids and vicinal fatty acid diols [17]. Although these biosynthetic pathways are well characterized in humans and other land-living mammals, little is known on eicosanoid biosynthesis in aquatic mammals. A PubMed search using the keywords 'marine mammals' and 'lipoxygenase' revealed some 70 hits but neither of these papers characterized the lipoxygenase pathway in marine mammals. In fact, to the best of our knowledge, it has never been explored systematically whether ALOX and COX isoforms do actually occur in aquatic mammals. To fill this gap, we first screened different publicly available sequence databases for ALOX-and COX-like sequences in marine mammals. Since we observed widespread occurrence of the two classes of enzymes in these animals, we selected species representing different subfamilies of marine mammals, for which complete ALOX15 sequences were available. These enzymes were expressed in pro-and eukaryotic overexpression systems and were characterized with respect to their catalytic properties. We found that all tested marine mammals express arachidonic acid 12-lipoxygenating ALOX15 orthologs and no 15-lipoxygenating enzymes could be identified. Thus, ALOX15 orthologs follow the Evolutionary Hypothesis of ALOX15 specificity. Since introduction of more space-filling amino acids at the triad positions altered the reaction specificity in favor of arachidonic acid 15-lipoxygenation, the enzymes follow the Triad concept.

Classification of marine mammals and their evolutionary relatedness
Mammals (Theria) are classified into three major subfamilies (Prototheria, Metatheria, and Eutheria), and Eutheria are subdivided into Xenarthra, Afrotheria, Euarchontoglires, and Laurasiatheria. Sea cows are marine mammals, which are classified as Afrotheria and these animals are closely related to the African Elephant. In contrast, most extant marine mammals are classified as Laurasiatheria. These animals originated from the northern supercontinent Laurasiatheria involve several land-living species such as shrews, even-toed and odd-toed ungulates, pangolins, and large predators, but also marine mammals, such as whales and dolphins. Moreover, the large predators also involve a number of marine mammals such as seals, walruses, sea bears, and polar bears. In Fig. 1, a systematic representation of different laurasiaterian marine mammals and their evolutionary relation is given. This image also summarizes those laurasiaterian marine mammals, which were selected for more detailed structural and functional investigations.

Database searches and sequence alignments of eicosanoid-synthesizing enzymes
Although eicosanoids are known as physiologically relevant lipid mediators for more than 50 years, little is known about the biological relevance of these compounds in marine mammals. To explore the eicosanoid-synthesizing capacity of different marine mammals, we first screened the publicly available sequence databases (protein databases, genome databases) for the presence of cyclooxygenase (COX1, COX2) and lipoxygenase (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) genes and judged the functionality of the corresponding enzymes identifying catalytically relevant amino acids.
Cyclooxygenases constitute key enzymes in the biosynthesis of prostaglandins, which have been implicated in a large number of physiological and pathophysiological processes [18][19][20][21]. The crystal structure of bovine COX1 has been solved [20], and dual amino acid sequence alignment with human COX1 identified catalytically important amino acids in the human ortholog (Table 1). Tyr384 is essential for the stereoselective hydrogen abstraction, which constitutes the rate-limiting step of the COX reaction. His206 (proximal) and His387 (distal) function as heme-binding ligands and, thus, are important for the peroxidase activity of this enzyme. COX1 is constitutively expressed in a large number of cells and tissues and is involved in the biosynthesis of physiological prostaglandins [18,21]. When we searched the public sequence databases (protein sequences and genomic sequences), we found that COX1 genes are present in all marine mammals tested (Table 1). Dual amino acid sequence alignments with human COX1 (Fig. S1a-n) indicated that the degree of amino acid identity of the identified COX1 orthologs with human COX1 varied between 50 and 90%. Moreover, in all sequences the catalytically important amino acids are conserved (Table 1) and these data suggested that the database sequences represent catalytically active enzymes.
COX2 is an inducible enzyme, which is expressed at high levels in stimulated inflammatory cells [18,19,21]. It is responsible for the biosynthesis of pro-inflammatory prostaglandins, and selective COX2 inhibitors have Cetacea and Canoidea represent two major subcategories of marine mammals, and we selected the species given on orange background for functional studies. *For the species marked with an asterisk, we retrieved complete ALOX15 sequences but the enzymes are not expressed in proand eukaryotic expression systems. **The ALOX15 sequence of the polar bear is incomplete, and thus, this enzyme could neither be expressed. However, the available sequences for these enzymes allowed prediction of the reaction specificity based on the Triad concept. been developed as potent anti-inflammatory drugs [22,23]. Functional COX2 genes were detected in all tested marine mammals, and the degree of amino acid identity with human COX2 varied between 70 and 90 % ( Table 2). Dual amino acid sequence alignments with human COX2 (Fig. S2a-n) indicated that in the extracted sequences, functionally important amino acids are conserved and these data suggest functionality of these genes and the corresponding enzymes.
ALOX15 is one of the six human ALOX isoforms. It has been implicated in cell differentiation and maturation [24][25][26][27] but also in the pathogenesis of inflammatory, hyperproliferative, and neurological disorders [16,28,29]. Its crystal structure is known [30,31], the enzyme has well been characterized, and catalytically important amino acids have been identified [32,33]. Primates, which are ranked in evolution above gibbons (humans, chimpanzees, orangutans, gorilla), express 15-lipoxygenating ALOX15 orthologs. In contrast, mammals ranked lower than gibbons in evolution express 12-lipoxygenating enzymes and gibbons represent the evolutionary turning point [34,35]. When we searched the sequence databases, we detected ALOX15 orthologs in all marine mammals tested. The Table 1. Presence of COX1 in different marine mammals. The amino acid sequences of the COX1 orthologs of different marine mammals were extracted from the protein database of the different mammals, and sequences were crosschecked using the corresponding genomic sequences. If required, corrections were introduced into the amino acid sequences based on the genomic data (*). In order to quantify the degree of amino acid conservation and to identify functionally relevant residues, dual amino acid sequence alignments were carried out with human COX1 (Fig. S1a-n). The deposited sequence of the polar bear and Weddell seal COX1 genes were incomplete (exons 1 + 2 are lacking), which might be related to a sequencing artifact.  Presence of COX2 in different marine mammals. The amino acid sequences of the COX2 orthologs of different marine mammals were extracted from the protein databases of the different mammals and from genome databases. Deposited protein sequences were crosschecked using the corresponding genomic sequences. *If required, minor corrections were introduced into the amino acid sequences based on the genomic data. In order to quantify the degree of amino acid conservation and to identify functionally relevant residues, dual amino acid sequence alignments were carried out with human COX2 (Fig. S2a- (Table 3), and the triad determinants involved the Val(Ala)418 + Val(Ala)419 motif ( Fig. S3a-n). These amino acid motifs are characteristic for arachidonic acid 12-lipoxygenating ALOX15 orthologs [35]. The Coffa determinant [33,36], which controls the stereochemistry of oxygen insertion, was an Ala in all cases ( Fig. S3a-n), and thus, all enzymes were predicted to function as arachidonic acid 12S-lipoxygenating enzymes. Human ALOX15B is an arachidonic acid 15-lipoxygenating enzyme [37] but its murine ortholog forms 8S-HETE as major arachidonic acid oxygenation product. As molecular reason for the altered reaction specificity of the two enzyme orthologs, a minor difference in the amino acid sequence has been identified [38]. Mutagenesis of the two amino acids located at positions 603 and 604 of mouse Alox15b (Jisaka determinants) to the corresponding amino acids present in human ALOX15B (His603Asp + His604Val) resulted in a 15S-lipoxygenating mutant, and the inverse mutagenesis strategy on human ALOX15B did also alter the reaction specificity [38]. The Triad concept is not applicable for mammalian ALOX15B orthologs [39]. Searching the sequence databases (protein and genomic sequences), we identified ALOX15B orthologs in all tested marine mammals (Table S1). Except for the polar bear, the iron ligands in ALOX15B are strongly conserved. Moreover, the Jisaka determinants were occupied by either the Asp + Val or the Asn + Ile motifs and the Coffa determinant was an Ala in all cases ( Fig. S4a-n). This sequence information suggested that the ALOX15B orthologs of marine mammals might function as Table 3. Presence of ALOX15 orthologs in different marine mammals. The amino acid sequences of ALOX15 orthologs of different marine mammals were extracted from the protein databases of the different mammals. These sequences were crosschecked by the corresponding genomic sequences, and if required, minor corrections were introduced into the amino acid sequences. In order to quantify the degree of amino acid conservation and to identify functionally relevant residues (iron ligands, triad determinants, stereo determinants), dual amino acid sequence alignments were carried out with human ALOX15 (Fig. S3a- arachidonic acid 15S-lipoxygenating enzymes. It should be stressed at this point that we did not express and functionally characterize any ALOX15B ortholog of marine mammals, and thus, this conclusion remains to be confirmed experimentally in subsequent expression studies. Mammalian ALOX12 orthologs oxygenate arachidonic acid to 12S-HETE [40], and this reaction specificity is consistent with the predictions made on the basis of the Triad concept [Phe + Val(Ala)+Val (Ala)+Ile(Val)] (Table S2). Except for the polar bear, the iron ligands are well conserved but this might be related to the observation that the polar bear ALOX12 is apparently 150 amino acids bigger than that of other marine mammals. In mice, knockout studies have implicated this enzyme in platelet function [41] and in epidermal differentiation [42], but recent studies also suggested a role in hemostasis [43]. When we searched the sequence databases (protein and genomic sequences), we found ALOX12 orthologs in all marine mammals tested (Table S2). The degree of amino acid identity with human ALOX12 varied between 53% and 83%. The triad determinants involve the Val (Ala)+Val(Ala) motif, which is characteristic for arachidonic acid 12-lipoxygenating enzymes, and an Ala occupies the Coffa position ( Fig. S5a-n). These data predict an arachidonic acid 12S-lipoxygenating enzyme for all tested marine mammals but experimental confirmation of this prediction is still pending.

Species
ALOX12B and ALOXE3 are two ALOX isoforms, which are high level expressed in the skin, and thus, they are frequently referred to as epidermal ALOX isoforms. Human and murine ALOX12B orthologs are arachidonic acid 12R-lipoxygenating enzymes [44,45], which have been implicated in epidermal differentiation. Systemic knockout of the Alox12b gene induced malfunction of the epidermal water barrier, and newborns die within hours after birth because of dehydration [46]. Mutations in the human ALOX12B gene have been related to the pathogenesis of congenital ichthyosis [47], a hereditary skin disease characterized by excessive scaling. The Triad concept is not applicable to ALOX12B orthologs [39], but the enantioselectivity of these enzymes (12R-lipoxygenating) is consistent with the observation that a Gly occupies the Coffa position [48]. When we screened the protein databases of marine mammals for ALOX12B orthologs, we could not find such sequences in any of the tested Cetaceaneae (whales and dolphins). In a second step, we searched the genomic sequences of these animals for ALOX12B genes using the human ALOX12B gene as probe. Here again, we did not find ALOX12B genes (Table S3), and taken together, these data indicate that the genomes of whales and dolphins do not involve functional ALOX12B genes. Cetaceans do not bear furs, and the structure of their skin is different when compared to that of land-living mammals, such as mice and humans. On the other hand (Table S3), we did detect functional ALOX12B orthologs in fur-bearing marine mammals (Weddell seal, northern fur seal, walrus, sea otter, polar bear) and these enzymes exhibit a high (70-80%) degree of amino acid identity with human ALOX12B. All iron ligands are conserved, and a Gly occupies the Coffa position ( Fig. S6a-e). These data suggest that these enzymes are catalytically active and that they might function as 12R-lipoxygenases. The lack of functional ALOX12B orthologs in Cetaceans might lead to the prediction that epidermal differentiation in these animals does not follow the mechanisms suggested for mice and humans [49,50].
ALOXE3 constitutes an epidermal ALOX isoform [51][52][53], which has been suggested to work in concert with ALOX12B during epidermal differentiation [49,50]. Systemic ALOXE3 deficiency also induces an ichthyosis-like phenotype in mice but the symptoms are less severe when compared to those observed for Alox12b knockouts [54]. Although recombinant ALOXE3 exhibits an arachidonic acid oxygenase activity (formation of racemic 5-HpETE, 7-HpETE, and 9-HpETE) in the presence of exogenous hydroperoxides and under hyperoxic conditions, it has been suggested to function in vivo as hydroperoxide isomerase [55][56][57]. When we explored the distribution of ALOXE3 orthologs in marine mammals (Table S4), we detected ALOXE3 genes in all tested species regardless of whether they are fur-bearing or not. After extraction of the cDNA sequences from the genomic data, we found that the degree of amino acid identity of these enzymes with human ALOXE3 varied between 70% and 75% and the iron ligands are conserved ( Fig. S7a-n). As in human ALOXE3, an Ala occupies the Coffa position (Table S4), and taken together, these data suggest the functionality of these enzymes. However, our dual amino acid sequence alignments also indicated the presence of premature stop codons and/or frameshifting point mutations in the coding region of the ALOXE3 genes of whales and dolphins, which interrupt their open reading frames. Based on these sequence data, one can conclude that although ALOXE3 genes are present in the genome of marine mammals, they do not encode for functional ALOXE3 orthologs in Cetaceaneae.
ALOX5 is the key enzyme in the biosynthesis of leukotrienes [58,59] and plays an important role in inflammatory diseases. The crystal structure of a  [60], and knockout mice show a selective opposition to certain inflammatory insults [61]. When fed a lipid-rich diet, these animals develop aortic aneurisms [62] suggesting a role of this enzyme in vascular remodeling [63]. Searching the protein databases, we detected ALOX5 sequences in all marine mammals (Table S5). The degree of amino acid identity with human ALOX5 varied between 88% and 95%, and the iron ligands are well conserved (Fig. S8a-n). Moreover, the triad determinants were identical with those amino acids identified at these positions in human ALOX5 and these data suggest the functionality of the corresponding enzymes. However, for the sperm whale we identified a truncated version of ALOX5 (412 amino acids instead of 674 amino acids), which lacks internal parts of the enzyme. Since the lacking sequences involve functionally relevant amino acids, it is most probable that this truncated ALOX5 version is catalytically silent. However, it might well be that the apparent truncation is a result of a sequencing artifact.

Expression of selected ALOX15 orthologs of marine mammals
To test the functionality of ALOX15 orthologs of different marine mammals and to compare the corresponding enzymes with those of land-living mammals, we selected different species representing distinct evolutionary subclasses (Fig. 1). Unfortunately, no sequence information is currently available for sea cows and other afrotherian marine mammals. In contrast, for laurasiatherian marine mammals a number of complete ALOX15 sequences are available and we selected the following species for expression and functional characterization ( Fig. 1  B-E) ALOX15 activity assays of different ALOX15 orthologs analyzed by RP-HPLC: Different volumes of the bacterial lysis supernatants were employed for the activity assays (see Materials and Methods) using arachidonic acid as substrate, and RP-HPLC analysis indicated the formation of specific ALOX15 products (12and 15-HETE). The chromatograms were scaled for the highest product peak (12-HETE). F) Activity assay of baiji ALOX15 analyzed by NP-HPLC: Arachidonic acid oxygenation products formed by recombinant baiji ALOX15 were prepared by RP-HPLC and further analyzed by NP-HPLC (see Materials and Methods). In this experiment, a modified cell lysis protocol was employed (see Materials and Methods). Here, we found the formation of specific lipoxygenase products by the ALOX15 orthologs of walrus (Fig. 2B), sea otter (Fig. 2C), Weddell seal (Fig. 2D), and the northern fur seal (Fig. 2E).
In these cases, the major oxygenation products comigrated in RP-HPLC with an authentic standard of 12-HETE. In addition, variable amounts of 15-HETE were also detected. No ALOX15 activity could be observed when the bacterial lysate supernatants of the beluga whale, the mink whale, and baiji were used as enzyme source. Together with the lacking western blot signals, these data suggest that the corresponding enzymes are either not well expressed in E. coli or that they have been deposited as denatured proteins in inclusion bodies. In fact, we observed large amounts of immunoreactive protein in the bacterial lysis pellets of mink whale and baiji. Although we performed a number of renaturation experiments, we could not restore the catalytic activity of these enzymes. No ALOX15 expression was observed for the ALOX15 ortholog of the beluga whale. To obtain functional data for baiji ALOX15, we employed a modified cell lysis protocol (see Materials and Methods). With this method, we obtained a catalytically active enzyme preparation and NP/CP-HPLC analysis of the oxygenation products revealed the formation of 12S-HETE as major oxygenation product (Fig. 2F). When we attempted to express the ALOX15 orthologs of the beluga whale and the mink whale in different eukaryotic overexpression systems (HEK293 cells, Sf9 insect cells), we never obtained sufficient amounts of catalytically active protein.
Finally, we tried to purify the recombinant enzymes by affinity chromatography on Ni-agarose. For some ALOX15 orthologs, this method worked very well and we obtained strongly enriched enzyme preparations. However, other ALOX15 orthologs could not be purified and we experienced a strong reduction of the catalytic activity. Thus, we decided not to purify the enzymes for more detailed catalytic characterization but use the lysate supernatants as enzyme source.

Functional characterization of recombinant ALOX15 orthologs
To compare the ALOX15 orthologs of marine mammals with human ALOX15, we quantified a number of enzyme characteristics, which have previously been determined for human ALOX15 [64]. Human ALOX15 is an arachidonic acid 15-lipoxygenating enzyme [65]. In contrast, the ALOX15 ortholog of the sea otter, which represents Mustelidae (Fig. 1), converts the same substrate mainly to 12-HETE (Fig. 2C). Small amounts of 15-HETE were also formed (Fig. 3A, right trace), and this dual reaction specificity is characteristic for mammalian ALOX15 orthologs [39,64,66,67]. NP-HPLC analysis (Fig. 3A, left trace) confirmed this finding, and chiral-phase HPLC indicated a strong preponderance of 12S-HETE over the corresponding R-enantiomer (Fig. 3A, middle trace).
Next, we determined the temperature dependence of arachidonic acid oxygenation by this enzyme and found a T opt of 15°C (Fig. 3B). At higher temperatures, the reaction rate declines suggesting a high degree of thermolability of the enzyme. From the data determined in the temperature range between 5°C and 15°C, we constructed the Arrhenius plot and calculated an activation energy of 32.3 kJÁmol À1 (Fig. 3C). The pH opt of arachidonic acid oxygenation was 8.0 (Fig. 3D), and a K M value of 26.9 µm was determined (Fig. 3E). Thus, the substrate affinity of the sea otter ALOX15 is comparable with that of the rabbit enzyme [68]. There was no significant increase in the reaction rate of arachidonic acid oxygenation (Fig. 3F) when the reaction was carried out under hyperoxic conditions, and these data suggest a high oxygen affinity of the enzyme.
Similar characterization studies were carried out for the ALOX15 orthologs of the northern fur seal (Fig. S9A-F) representing Otariidae (Fig. 1), Weddell seal ( Fig. S10A-F) representing Phocidae (Fig. 1), and walrus ( Fig. S11A-F) representing Odobenidae (Fig. 1). The kinetic parameters are summarized in Table 4. Unfortunately, there is no sufficient sequence information currently available in the databases to express and characterize the ALOX15 ortholog of the polar bear representing Ursinae (Fig. 1).
Membrane oxygenase activity of the ALOX15 orthologs of marine mammals ALOX15 orthologs of different mammals exhibit biomembrane oxygenase activity [69][70][71]. To explore whether the ALOX15 orthologs of marine mammals are also capable of oxygenating membrane-bound polyenoic fatty acids, we incubated the recombinant enzyme preparations with mitochondrial membranes (submitochondrial particles, SMP) and analyzed by HPLC the oxygenation products in the hydrolyzed lipid extracts. From Fig. 4C, it can be seen that conjugated dienes are present in the hydrolyzed lipid extracts of SMPs treated with the ALOX15 ortholog of the northern fur seal. In no-enzyme (membranes only) controls, the conjugated diene peak was much   (Fig. 4D) and the no-enzyme control ( Fig. 4B) look very similar, and these data suggest that ALOX15 incubation does not oxygenate a large share of the polyenoic fatty acids. To quantify the degree of oxidation of the membrane lipids, we next determined the amounts of conjugated dienes (OH-PUFAs) and the amounts of polyenoic fatty acids (PUFAs) present in the hydrolyzed lipid extracts prepared from the incubations with ( Fig. 4C + D) and without ( Fig. 4A + B) ALOX15 treatment and calculated the OH-PUFA/PUFA ratios. Here, we found that 1.72% of the two major PUFAs were present as oxygenated derivatives after ALOX15 treatment. Similar values were obtained when these membranes were treated with native rabbit ALOX15 [69], recombinant human ALOX15 [64], or recombinant rat ALOX15 [67]. Identical experiments were carried out for the ALOX15 orthologs of Weddell seal, sea otter, and walrus, and the analytical data are summarized in Table 5. To further characterize the major oxygenation products, we pooled the products eluted in the HODE/HETE region of the four different ALOX15 orthologs, prepared the conjugated dienes formed, and analyzed them by combined normal-phase/chiral-phase (NP/CP-) HPLC. Here, we confirmed (Fig. 4E) that esterified 13-HODE and 12-HETE were the major conjugated dienes. Similar product patterns were identified when ALOX15 orthologs of other marine mammals were used as catalysts (Table 6).
Taken together, these data indicate that the ALOX15 orthologs of marine mammals exhibit membrane oxygenase activity and that esterified 13-HODE and 12-HETE are the major oxygenation products, when these enzymes oxygenate mitochondrial membrane lipids.

Mutagenesis studies on the triad determinants
Human ALOX15 is an arachidonic acid 15-lipoxygenating enzyme [72], whereas the mouse [73] and rat orthologs [67,74] are 12-lipoxygenating. The Triad concept explaining the variable reaction specificity of mammalian ALOX15 orthologs suggests that the side chain geometry of the triad determinants (Phe353, Ile418 + Met419, Ile593 of human ALOX15) is important for the reaction specificity. When small amino acids are located at the positions, which align with these amino acids of human ALOX15, the corresponding enzyme exhibits an arachidonic acid 12-lipoxygenating activity. In contrast, when more space-filling side chains are localized at these positions, the formation of 15-HETE is dominant [29]. To explore whether the ALOX15 orthologs of marine mammals follow the Triad concept, we first carried out an amino acid sequence alignments with human ALOX15 and identified the triad determinants ( Fig. S3a-n). Here, we found that Phe353 (Borngraber determinant I) of human ALOX15 is strongly conserved in marine mammals. In contrast, at the Sloane positions (Ile418 + Met419 of human ALOX15) two small amino acids (Val or Ala) are located in marine mammals (Fig. S3a-n). These data suggest that the ALOX15 orthologs of marine mammals function as arachidonic acid 12-lipoxygenating enzymes and this conclusion is consistent with the results of our activity assays (Fig. 2). To test whether introduction of more space-filling residues at the triad determinants alters the reaction specificity of these enzymes in favor of arachidonic acid 15-lipoxygenation, we first expressed the following mutants of the northern fur seal ALOX15: Val418Ile, Val419Met, and Val418Ile + Val419Met. The analysis of the arachidonic acid oxygenation products is shown in Fig. 5.
The wild-type enzyme is a major 12-lipoxygenating enzyme (Fig. 2), and we confirmed this finding in our mutagenesis studies (Fig. 5). The Val418Ile mutant exhibits pronounced dual reaction specificity since similar amounts of 12-and 15-H(p)ETE are formed (Fig. 5). The Val419Met mutant prefers the formation of 12-H(p)ETE but compared with the wild-type enzyme, the share of 15-H(p)ETE formation was clearly augmented (Fig. 5). The Val418Ile + Val419Met double mutant functions as dominant 15-lipoxygenating enzyme, and the share of 12-H(p)ETE formation dropped down to about 10%.
The Triad concept also predicts that alterations in the reaction specificity induced by mutations of the Sloane determinants (Val418 + Val419) can be reversed, when the Borngraber I determinant (Phe353) is mutated [32]. To find out whether this is also the case for the ALOX15 ortholog of the northern fur seal, we mutated in the Val418Ile + Val419Met double-mutant Phe353 to smaller amino acids (Leu, Ala). We found that the Phe353Leu + Val418Ile+Val419Met and the Phe353Ala + Val418Ile+Val419Met triple mutants catalyzed almost exclusive arachidonic acid 12-lipoxygenation (Fig. 5). Thus, introduction of less space-filling residues at Phe353 completely inverted the alterations in reaction specificity induced by Val418Ile + Val419Met exchange. Similar results were obtained when the ALOX15 orthologs of sea otter, walrus, and Weddell seal were taken through this experimental protocol (Table 7). Taken together, these data indicate that the Borngraber-1 (Phe353) determinant and the Sloane determinants (Val/Ala418 + Val/Ala419) functionally interact with each other and impact the reaction specificity of these enzymes in a concerted way. From these data, one can conclude that the Triad concept is applicable to ALOX15 orthologs of all marine mammals included in this study.

Eicosanoid-synthesizing enzymes in marine mammals
Eicosanoids are lipid mediators that have been implicated in homeostasis but also in the pathogenesis of important diseases. COX-inhibitors are potent anti-inflammatory drugs [22], and leukotriene receptor (E) Normal-phase (NP-) HPLC analysis of the conjugated dienes formed during the membrane oxygenase activity assays carried out with the ALOX15 orthologs of Weddell seal, northern fur seal, sea otter, and walrus: Membrane oxygenase activity assays were performed with the ALOX15 orthologs of these marine mammals (see Materials and Methods). The conjugated dienes were prepared by RP-HPLC and pooled together. Solvent was evaporated; lipids were reconstituted in normal-phase HPLC solvent and analyzed by NP-HPLC (see Materials and Methods). Inset: The UV spectra of the products co-eluting with authentic standards of 12-HETE and 13-HODE. The peak marked with an asterisk (*) did not exhibit a typical diene chromophore, and control preparations indicated that it originated from the RP-HPLC solvent. antagonists [75] are available for prescription as antiasthmatics. Despite our comprehensive knowledge on eicosanoid biology in mice and humans, little is known on the role of these mediators in marine mammals. In this study, we systematically searched the publicly available protein databases for eicosanoid-synthesizing enzymes in marine mammals and found that COX1, which is responsible for the biosynthesis of physiological prostaglandins, is present in all tested marine mammals (Table 1). According to the results of our sequence alignments, the enzymes detected should be functional since the functionally important amino acids are well conserved. COX2 orthologs have also been detected in all marine mammals tested ( Table 2). Here again, the functional amino acids are strongly conserved suggesting catalytic activity of the corresponding enzymes.
ALOX15 orthologs have been detected in all marine mammals tested (Table 3), and these data indicate a high degree of distribution of this enzyme in these animals. As for COX2, catalytically important amino acids (iron ligands, triad determinants, stereo determinants) are well conserved, and thus, the enzymes should be catalytically active. This conclusion is supported by the catalytic activity of the recombinant ALOX15 enzymes expressed in our study. Other ALOX isoforms (ALOX15B, ALOX12, ALOX12b, ALOXE3, ALOX5) were also detected in most marine mammals (Table S1-S5), and here again, the catalytically important amino acids are conserved. Interestingly (Table S3), we did not find ALOX12B genes in any of the nine Cetacean species  (whales and dolphins) tested and this conclusion was based on the data obtained screening the protein databases and genomic sequences of these animals. Cetaceae do not carry a fur, and the skin of these animals is differently structured when compared to fur-bearing mammals. Interestingly, ALOX12B orthologs were present in all tested fur-bearing marine mammals (Table S3) and these data suggest that this enzyme may be related to fur development. ALOX12B and ALOXE3 have previously been implicated in epidermal differentiation [47,49,50] in mice and humans. In fact, Alox12b knockout mice die from dehydration shortly after birth [76], and thus, the enzyme has been implicated in the formation of the epidermal barrier preventing water evaporation. Since Cetaceaneae are born in the water and spend their entire life in aqueous surroundings, they do not need an epidermal water evaporation barrier. Thus, there might be no evolutionary pressure on the ALOX12B genes in Cetaceaneae, and the lack of such pressure might have led to the disappearance of these genes. Interestingly, ALOXE3 genes are present in the genomes of the tested Cetaceaneae (Table S4). This finding was rather surprising, since ALOX12B and ALOXE3 orthologs have been implicated in the formation of the epidermal water barrier. However, when we explored the genomic ALOXE3 sequences of Cetaceaneae in more detail we detected premature stop codons and/or frameshifting point mutations in the coding regions of the corresponding genes (Fig. S7) and these data suggest that these genes do not encode for functional proteins. Thus, according to these sequence data Cetaceaneae may not express functional ALOXE3 orthologs.
Comparison of ALOX15 orthologs of marine mammals with those of land-living mammals In general, ALOX15 orthologs exhibit a broad substrate specificity accepting most naturally occurring polyenoic fatty acids as substrate. The ALOX15 orthologs of marine mammals oxygenate arachidonic acid to 12S-HETE (Fig. 2), and 13S-HODE was the major product of linoleic acid oxygenation (data not shown). When the two fatty acids were simultaneously supplied as constituents of biomembranes, 13S-HODE formation was dominant (Fig. 4).
The K M value for arachidonic acid oxygenation varied between 44 and 160 µm for the different marine mammalian ALOX15 orthologs tested. These data values are higher than the K M values determined for native rabbit and recombinant human ALOX15 for linoleic acid [64,68]. It should, however, been stressed that we assayed the fatty acid oxygenase activity in the absence of any detergent, which lowers the water solubility of the substrate fatty acids and, thus, its availability for the enzymes. In a similar experimental setup (no detergent), we determined similar K M values for arachidonic acid for the ALOX15 orthologs of P. aeruginosa [71] and T. belangeri [77].
Compared with the ALOX15 orthologs of land-living mammals, the thermostability of the enzymes of marine mammals is rather low. At room temperature, the enzymes undergo rapid inactivation and even on ice the enzyme preparations loose catalytic activity. This catalytic instability might also be discussed as reason why the enzymes inactivate during affinity chromatography purification. The molecular basis for the catalytic instability has not been explored.
Mammalian ALOX and COX isoforms exhibit a high affinity for atmospheric oxygen, and the oxygen K M varied between 10 and 26 µm [78]. In contrast [79], oxygen affinity of the 15-lipoxygenating P. aeruginosa ALOX was much lower (K M of 0.4 mM). When we studied the impact of higher oxygen concentrations on the rate of arachidonic acid oxygenation, we did not observe increased rates under hyperoxic conditions (Fig. 3, Fig. S9, S10, S11). Although we did not exactly quantify the oxygen K M , the lacking increase Table 7. Applicability of the Triad concept for ALOX15 orthologs of marine mammals. ALOX15 orthologs of marine mammals were mutated, and the single, double, and triple mutants were employed for activity assays (see Materials and Methods). The major oxygenation products were identified by RP-HPLC strategy. The 12S-HETE/15S-HETE ratios are given in the in catalytic activity under hyperoxia suggests that under normoxic conditions, the enzymes are oxygen saturated. Thus, these data suggest a high oxygen affinity of the ALOX15 orthologs of marine mammals. All tested ALOX15 orthologs of marine mammals are capable of oxygenating membrane ester lipids (Fig. 4, Table 5). This catalytic activity was first described for rabbit ALOX15 [80] and has later been confirmed for the enzyme orthologs of other land-living mammals [64,67,70]. Unfortunately, for ALOX15 orthologs of marine mammals no information on this catalytic property has been available. Here, we showed that the tested ALOX15 orthologs of marine mammals exhibit a membrane oxygenase activity but compared with the fatty acid oxygenase activity this catalytic activity was 10-fold less efficient. This finding is consistent with the observations made for other mammalian ALOX15 orthologs [81]. Although the mechanistic basis for this phenomenon has not been explored, it may be possible that the membrane proteins protect the membrane lipids from the ALOX15 attack.

The ALOX15 orthologs of marine mammals follow the Triad concept
The Triad concept of ALOX15 specificity suggests that three clusters of amino acids (triad determinants) are important for the reaction specificity [32]. In human and rabbit ALOX15, these determinants are Phe353 (Borngraber-1 determinant), Ile418 + Met419 (Sloane determinant), and Ile593 (Borngraber-2 determinant) [32,82,83]. In all marine mammalian ALOX15 orthologs, a Phe aligns with Phe353 of human ALOX15 (Fig. S3a-n). Ile418 + Met419 of human ALOX15 aligns with Val + Val, Val + Ala, or Ala + Val motifs in marine mammalian ALOX15 orthologs. Thus, small amino acids are present at the Sloane determinants. Ile593 of human ALOX15 is conserved in all marine mammalian ALOX15 orthologs. Taken together, these sequence data predict arachidonic acid 12-lipoxygenating ALOX15 orthologs for all marine mammals. To explore whether the triad determinants functionally interact with each other, we introduced the more bulky amino acids present in human ALOX15 at these positions into the marine enzyme orthologs and observed a gradual change in the reaction specificity favoring arachidonic acid 15-lipoxygenation (Table 7). In fact, the Val418Ile + Val419Met double mutants were dominantly 15lipoxygenating. When we introduce smaller amino acids at the Borngraber-1 position (Val418Ile + Val419Met+ Phe353Leu, Val418Ile + Val419Met+Phe353Ala), the changes induced by mutagenesis of the Sloane determinants (Val418Ile + Val419Met) were reversed and dominantly 12-lipoxygenating triple mutants resulted (Fig. 5). Taken together, these data indicate that the Triad concept explains the reaction specificity of the ALOX15 orthologs of marine mammals and that the triad determinants functionally interact with each other.
The reaction specificity of the ALOX15 orthologs of marine mammals supports the Evolutionary Hypothesis of ALOX15 specificity The Evolutionary Hypothesis of ALOX15 specificity suggests that mammals ranked in evolution above gibbons (humans, chimpanzee, gorillas, orangutans) express an arachidonic acid 15-lipoxygenating ALOX15. In contrast, lower primates and other mammals express arachidonic acid 12-lipoxygenating enzymes [35,84]. There are rare exceptions, but more than 90 % of all functionally characterized mammalian ALOX15 orthologs follow this concept [35,84]. If this hypothesis is applicable for marine mammals, their ALOX15 orthologs should function as arachidonic acid 12-lipoxygenating enzymes. Analysis of the sequence data we extracted from the databases ( Table 3) indicates that the Sloane determinants are occupied by small amino acids (Val/Ala418, Val/ Ala419), and on the basis of the Triad concept [35], one can predict arachidonic acid 12-lipoxygenating activity for the 14 marine mammal ALOX15 orthologs we found in the database. Four of them have been expressed as recombinant enzyme and functional characterization confirmed this prediction (Fig. 3). Thus, the reaction specificities of the functionally tested ALOX15 orthologs of marine mammals support the Evolutionary Hypothesis.

Chemicals
The chemicals were purchased from the following vendors: acetic acid from Carl Roth GmbH (Karlsruhe, Germany); sodium borohydride from Life Technologies, Inc. (Eggenstein, Germany); antibiotics and isopropyl-b-thiogalactopyranoside (IPTG) from Carl Roth GmbH (Karlsruhe, Germany); restriction enzymes from Thermo Fisher Scientific-Fermentas (Schwerte, Germany); the E. coli strain Rosetta2 DE3 pLysS from Novagen (Merck-Millipore, Darmstadt, Germany); and E. coli strain XL-1 from Stratagene (La Jolla, CA, USA). Oligonucleotide synthesis was performed at BioTez Berlin Buch GmbH (Berlin, Germany). Nucleic acid sequencing was carried out at Eurofins Genomics Germany GmbH (Ebersberg, Germany). HPLCgrade methanol, acetonitrile, and water were purchased In silico database searches and sequence alignments Initially, we extracted the amino acid sequences of ALOX and PGHS orthologs of marine mammals from the public protein database (https://www.ncbi.nlm.nih.gov/protein) using the key words 'lipoxygenase' and 'prostaglandin H synthase'. In a second step, we searched the genomic sequences deposited in the genome databases of the different marine mammals using the genomic sequence of human PGHS and ALOX genes. The genomes of all the marine mammals explored in this study have been sequenced completely by next-generation sequencing techniques, and the different sequence reads were properly assembled. The genome coverage of the assemblies ranges from to 27-fold to 248-fold and, thus, were of high quality. The data are accessible to the public and can be downloaded from the National Center for Biotechnology Information (https:// www.ncbi.nlm.nih.gov/assembly/). The BLAST 2.10.0 + standalone software package was used for searching ALOXand PGHS-like sequences in the genomes and to process the downloaded data. Initially, the makeblastdb program that converts fasta files to BLAST database files was used for creating a separate database for each species. Speciesspecific ALOX-and PGHS-like sequences were identified by screening each organism database using the corresponding sequences of the orthologous human genes employing the tblastn/blastn programs. The sequence hits identified were then analyzed on DNASTAR Lasergene (DNAS Inc., Madison, WI, USA), and the complete PGHS and ALOX sequences for each organism were identified. From the genomic sequences, we extracted the amino acid sequences and the protein database sequences were corrected for the genomic sequences if differences were observed. For amino acid alignments, we used the online tool https://www.eb i.ac.uk/Tools/psa/emboss_needle/.

Cloning of ALOX15 orthologs of marine mammals
To test the functionality of selected ALOX15 orthologs of marine mammals, we first extracted the cDNAs from the genomic sequence and optimized the coding regions for prokaryotic expression. Single amino acids missing in the extracted coding sequences were replaced by comparison with the sequences of the next relatives (mink whale, baiji, beluga). A SalI restriction site was introduced immediately upstream of the starting ATG of the his-tag fusion construct, and a HindIII recognition sequence was designed downstream of the stop codon. Internal HindIII and SalI sites were eliminated by silent nucleotide exchanges, and the construct was chemically synthesized (BioCat GmbH, Heidelberg, Germany). For prokaryotic expression, the construct was excised from the synthesizing vector (pUC57) and cloned into the expression plasmid pET28b (Novagen/ Merck, Darmstadt, Germany). Recombinant expression plasmids were tested for the ALOX15 inserts by SalI + HindIII digestion, and the final expression constructs were sequenced (Eurofins Genomics Germany GmbH, Ebersberg, Germany).

Bacterial expression of ALOX15 orthologs
Competent bacteria (Rosetta 2 DE3 pLysS) were transformed with 100ng of recombinant expression plasmids, and the cells were grown overnight on kanamycin/chloramphenicol containing agar plates. An isolated growing clone was selected and two 1 mL bacterial liquid cultures (LB medium with 50 lgÁmL À1 kanamycin/35 µgÁmL À1 chloramphenicol) were grown at 37°C. This preculture was added to a 50 mL main culture, and the cells were grown to reach an optical density (A 600 ) of 0.10-0. 15

Eukaryotic expression of ALOX15 orthologs in Sf9 cells
The expression was performed as described [84]. Briefly, the coding sequence was ligated into the pFastBac HT vector and the bacmid as well as the recombinant baculovirus

Site-directed mutagenesis
About 10-50 ng of plasmid DNA was incubated with the specific primer pair (1 µL of 5 µm solution each) and 12.5 µL of Pfu Ultra II Hot Start 2 9 PCR Master Mix (Agilent Technologies, Waldbronn, Germany) in a total volume of 25 µL adjusted with sterile water. The PCR protocol was designed as follows: 95°C for 1-min initial denaturation, cycle: 95°C for 30s > 55°C for 60s> 68°C for 10 min; altogether, 18 cycles were applied. Subsequently, the parent DNA was digested with 1 µL DpnI (Thermo Scientific, Schwerte, Germany) for 30min, followed by denaturation at 80°C for 10 min. About 8 µL of the PCR sample was used for transformation of 100 µL of E. coli XL-1 Blue competent cells (Agilent Technologies Inc., Santa Clara, USA). After incubation for 30 min on ice, the cells were heat-shocked for 45 s at 42°C and kept on ice for 2 min, and then, 400 µL of SOC medium was added. After 1-h incubation at 37°C and 180 rpm, the cells were plated on an LB-agar plate supplemented with 50 µgÁmL À1 kanamycin (for pET28b) or 100 µgÁmL À1 ampicillin (for pFastBac HT) and incubated overnight at 37°C. Four clones were selected for liquid culture in 2 mL of LB medium, and plasmid DNA was prepared using the NucleoSpin Plasmid Kit (Macherey & Nagel, D€ uren, Germany). One clone was selected for sequencing (Eurofins Genomics Germany GmbH, Ebersberg, Germany).

Activity assays and HPLC analysis
To assay the catalytic activity of the recombinant ALOX15 orthologs, variable amounts of the bacterial lysis supernatants were added to 0.5 mL of PBS containing 100 µm arachidonic acid as substrate. After 8 min of incubation, the hydroperoxy fatty acids were reduced by the addition of 1 mg of solid sodium borohydride, and after 5 min, the reaction mixture was acidified with 45 µL of concentrated acetic acid. Then, the proteins were denatured by the addition of 0.5 mL acetonitrile and precipitates were removed by centrifugation. Aliquots (50-300 µL) of the clear supernatants were injected to RP-HPLC analyses on a Shimadzu instrument, which was connected to a Hewlett Packard diode array detector 1040 A. Metabolites were separated on a Nucleodur C18 Gravity column (Macherey-Nagel, D€ uren, Germany; 250 9 4 mm, 5 lm particle size) coupled with a corresponding guard column (8 9 4 mm, 5 lm particle size). A solvent system consisting of acetonitrile:water: acetic acid (70 : 30 : 0.1, by vol) was used at a flow rate of 1 mLÁmin À1 . For more detailed analysis, the conjugated dienes were prepared by RP-HPLC and further analyzed by normalphase HPLC (NP-HPLC) and/or chiral-phase HPLC (CP-HPLC). Normal-phase HPLC was performed on a Nucleosil 100-5 column (250 9 4.6 mm, 5 µm particle size) using the solvent system n-hexane/2-propanol/acetic acid (100/2/0.1, by volume) at a flow rate of 1 mLÁmin À1 . 12-HETE enantiomers were resolved on a Chiralpak AD-H column (Daicel Corp., Osaka, Japan) with a solvent system consisting of n-hexane/methanol/ethanol/acetic acid (96 : 3 : 1 : 0.1, by vol, 1 mLÁmin À1 ).

Membrane oxygenase activity
To test the membrane oxygenase activity of the ALOX15 orthologs, we incubated different volumes of the bacterial lysis supernatants in 0.5 mL PBS with submitochondrial particles (1.4 mgÁmL À1 final membrane protein concentration). After 15 min, the reaction was stopped by the addition of 1 mg of sodium borohydride. Then, the sample was acidified with acetic acid, total lipids were extracted [86], and the ester lipids were hydrolyzed. After acidification, aliquots of the hydrolysates were analyzed by RP-HPLC and the chromatograms were followed simultaneously at 235 nm (conjugated dienes) and at 210 nm (nonoxidized polyenoic fatty acids). Quantifying the peak areas of the Federation of European Biochemical Societies major polyenoic fatty acids (LA + AA) and the conjugated dienes formed, we calculated the hydroxy PUFA/PUFA ratio, which constitutes a suitable measure for the degree of oxygenation of the membrane ester lipids [69].

Statistics
Statistical calculations and figure design were performed using MS Excel 2016 or GRAPHPAD PRISM version 8.00 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Table S1. Presence of ALOX15B orthologs in different marine mammals. Table S2. Presence of ALOX12 orthologs in different marine mammals. Table S3. Presence of ALOX12B orthologs in different marine mammals. Table S4. Presence of ALOXE3 orthologs in different marine mammals. Table S5. Presence of ALOX5 orthologs in different marine mammals. Figure S1. Amino acid alignments of COX1 orthologs of different marine mammals with human COX1. Figure S2. Amino acid alignments of COX2 orthologs of different marine mammals with human COX2. Figure S3. Amino acid alignments of ALOX15 orthologs of different marine mammals. Figure S4. Amino acid alignments of ALOX15B orthologs of different marine mammals. Figure S5. Amino acid alignments of ALOX12 orthologs of different marine mammals. Figure S6. Amino acid alignments of ALOX12B orthologs of different marine mammals. Figure S7. Amino acid alignments of ALOXE3 orthologs of different marine mammals. Figure S8. Amino acid alignments of ALOX5 orthologs of different marine mammals. Figure S9. Characterization of basic catalytic properties of the northern fur seal ALOX15. Figure S10. Characterization of basic catalytic properties of the Weddell seal ALOX15. Figure S11. Characterization of basic catalytic properties of the walrus ALOX15.