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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.

Lipoxygenases (ALOX-isoforms) are lipid peroxidizing enzymes, which have been implicated in cell differentiation and maturation but also in the biosynthesis of lipid mediators playing important roles in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. In mammals these enzymes are widely distributed and the human genome involves six functional genes encoding for six distinct human ALOX paralogs. In mice, there is an orthologous enzyme for each human ALOX paralog but the catalytic properties of human and mouse ALOX orthologs show remarkable differences. ALOX inhibitors are frequently employed for deciphering the biological role of these enzymes in mouse models of human diseases but owing to the functional differences between mouse and human ALOX orthologs the uncritical use of such inhibitors is sometimes misleading. In this study we evaluated the paralog- and ortholog-specificity of 13 frequently employed ALOX-inhibitors against four recombinant human and mouse ALOX paralogs (ALOX15, ALOX15B, ALOX12, ALOX5) under different experimental conditions. Our results indicated that except for zileuton, which exhibits a remarkable paralog-specificity for mouse and human ALOX5, no other inhibitor was strictly paralog specific but some compounds exhibit an interesting ortholog-specificity. Because of the variable isoform specificities of the currently available ALOX inhibitors care must be taken when the biological effects of these compounds observed in complex in vitro and in vivo systems are interpreted.

Endocannabinoids, such as anandamide (ANA) and 2-arachidonoylglycerol (2AG), are lipid-signaling molecules that can be oxidized by lipid-peroxidizing enzymes, and this oxidation alters the bioactivity of these lipid mediators. Here, under strictly comparable experimental conditions, we explored whether ANA and 2AG function as substrates for four human (ALOX15, ALOX15B, ALOX12, ALOX5) and three mice Alox isoforms (Alox15, Alox12, Alox5) and compared the rates of product formation with those of arachidonic acid oxygenation. Except for ALOX5, the two endocannabinoids were more efficiently oxygenated than arachidonic acid by human ALOX isoforms. Mice Alox15 oxygenated ANA more efficiently than arachidonic acid, but the other mice Alox isoforms exhibited reduced reaction rates for endocannabinoid conversion. Like its human ortholog, mice Alox5 did not oxygenate ANA, but the formation of 5-HETE-containing 2AG derivatives was observed for this enzyme. 1AG and 2AG were similarly effective substrates for human ALOX isoforms. Molecular docking studies, the pattern of oxygenation products, and site-directed mutagenesis experiments suggested a similar substrate alignment of arachidonic acid and endocannabinoids at the active site of ALOX15 orthologs. The product specificity of arachidonic acid oxygenation was conserved for endocannabinoid metabolization, and the triad concept describing the molecular basis for the reaction specificity of ALOX15 orthologs is applicable for endocannabinoid oxygenation. Taken together, these data indicate that, except for ALOX5 orthologs, endocannabinoids are suitable substrates for most mammalian ALOX isoforms.

Recent developments in the dual inhibition studies of cyclooxygenase-2 (COX-2) and carbonic anhydrase (CA-II) imply a promising platform for the development of new generations of nonsteroidal anti-inflammatory drugs (NSAIDs). CG100649 is such a molecule that got recently approved by Korean Ministry of Food and Drug safety (MFDS) and is being marketed by the name polmacoxib for the treatment of osteoarthritis. CG100649 significantly inhibits CA-II in blood and COX-2 in inflammatory tissues. However, the mechanism of CG100649 dual inhibition of COX-2/CA-II is not well understood. In this study, we employed well known methods like pharmacophore modelling, a DFT based quantum chemical descriptors analysis, and molecular docking to explore the chemical features and to understand the binding behaviour of CG100649 along with other COX-2/CA-II dual inhibitors. The HOMO-LUMO and docking results indicated the prominent role of aryl sulphonamide in CG100649. The aryl sulphonamide moiety formed T-shaped Π…Π interactions with His94 in the CA-II active site, which was not observed in the case of celecoxib. Other crucial interactions were also observed which may aid in further understanding the action of dual inhibitors of this class.

Mammalian lipoxygenases (LOXs) have been implicated in cell differentiation and in the biosynthesis of pro- and anti-inflammatory lipid mediators. The initial draft sequence of the Homo neanderthalensis genome (coverage of 1.3-fold) suggested defective leukotriene signaling in this archaic human subspecies since expression of essential proteins appeared to be corrupted. Meanwhile high quality genomic sequence data became available for two extinct human subspecies (H. neanderthalensis, Homo denisovan) and completion of the human 1000 genome project provided a comprehensive database characterizing the genetic variability of the human genome. For this study we extracted the nucleotide sequences of selected eicosanoid relevant genes (ALOX5, ALOX15, ALOX12, ALOX15B, ALOX12B, ALOXE3, COX1, COX2, LTA4H, LTC4S, ALOX5AP, CYSLTR1, CYSLTR2, BLTR1, BLTR2) from the corresponding databases. Comparison of the deduced amino acid sequences in connection with site-directed mutagenesis studies and structural modeling suggested that the major enzymes and receptors of leukotriene signaling as well as the two cyclooxygenase isoforms were fully functional in these two extinct human subspecies.

5-Lipoxygenase (ALOX5) is a key enzyme in biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Although mammalian LOX-isoforms share a high degree of structural similarity X-ray coordinates indicated that the substrate-binding pocket of ALOX5 is some 20% bigger than that of ALOX15 suggesting the possibility of interconverting the two isoenzymes. To test this "space-based" hypothesis we reduced the volume of the substrate-binding pocket of mouse Alox5 by introducing space-filling amino acids at critical positions and found that multiple mutations at Phe359, Ala424, Asn425 and Ala603 of Alox5 led to gradual increase in 15-HETE formation. The Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant was a major (67 ± 2%) 15-lipoxygenating enzyme and similar data were confirmed for human ALOX5. Structural modeling on the basis of the X-ray coordinates of ALOX5 indicated that the volume of the substrate-binding pocket inversely correlates with the share of 15-HETE biosynthesis for the human (r(2) = 0.79, p0.05) and the mouse (r(2) = 0.59, p0.01) enzyme. This data proves the principle possibility of converting pro-inflammatory 5-lipoxygenases to anti-inflammatory 15-lipoxygenases by reducing the volume of the substrate-binding pocket.

Leukotrienes are pro-inflammatory lipid mediators, which are biosynthesized via the lipoxygenase pathway of the arachidonic acid cascade. Lipoxygenases form a family of lipid peroxidizing enzymes and human lipoxygenase isoforms have been implicated in the pathogenesis of inflammatory, hyperproliferative (cancer) and neurodegenerative diseases. Lipoxygenases are not restricted to humans but also occur in a large number of pro- and eucaryotic organisms. Lipoxygenase-like sequences have been identified in the three domains of life (bacteria, archaea, eucarya) but because of lacking functional data the occurrence of catalytically active lipoxygenases in archaea still remains an open question. Although the physiological and/or pathophysiological functions of various lipoxygenase isoforms have been studied throughout the last three decades there is no unifying concept for the biological importance of these enzymes. In this review we are summarizing the current knowledge on the distribution of lipoxygenases in living single and multicellular organisms with particular emphasis to higher vertebrates and will also focus on the genetic diversity of enzymes and receptors involved in human leukotriene signaling.

Mammalian lipoxygenases play a role in normal cell development and differentiation but they have also been implicated in the pathogenesis of cardiovascular, hyperproliferative and neurodegenerative diseases. As lipid peroxidizing enzymes they are involved in the regulation of cellular redox homeostasis since they produce lipid hydroperoxides, which serve as an efficient source for free radicals. There are various epidemiological correlation studies relating naturally occurring variations in the six human lipoxygenase genes (SNPs or rare mutations) to the frequency for various diseases in these individuals, but for most of the described variations no functional data are available. Employing a combined bioinformatical and enzymological strategy, which included structural modeling and experimental site-directed mutagenesis, we systematically explored the structural and functional consequences of non-synonymous genetic variations in four different human lipoxygenase genes (ALOX5, ALOX12, ALOX15, and ALOX15B) that have been identified in the human 1000 genome project. Due to a lack of a functional expression system we resigned to analyze the functionality of genetic variations in the hALOX12B and hALOXE3 gene. We found that most of the frequent non-synonymous coding SNPs are located at the enzyme surface and hardly alter the enzyme functionality. In contrast, genetic variations which affect functional important amino acid residues or lead to truncated enzyme variations (nonsense mutations) are usually rare with a global allele frequency, Highlights • Non-synonymous coding variations in human lipoxygenases are mostly rare with a global allele frequency

Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity.