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

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.

Secreted lipoxygenase from Pseudomonas aeruginosa exhibits biomembrane oxygenase activity and induces hemolysis in human red blood cells.

Pseudomonas aeruginosa (PA) expresses a secreted lipoxygenase (LOX), which oxygenates free arachidonic acid predominantly to 15S-H(p)ETE. The enzyme is capable of binding phospholipids at its active site and physically interacts with model membranes. However, its membrane oxygenase activity has not been quantified. To address this question, we overexpressed PA-LOX as intracellular his-tag fusion protein in Escherichia coli, purified it to electrophoretic homogeneity and compared its biomembrane oxygenase activity with that of rabbit ALOX15. We found that both enzymes were capable of oxygenating mitochondrial membranes to specific oxygenation products and 13S-H(p)ODE and 15S-H(p)ETE esterified to phosphatidylcholine and phosphatidylethanolamine were identified as major oxygenation products. When normalized to similar linoleic acid oxygenase activity, the rabbit enzyme exhibited a much more effective mitochondrial membrane oxygenase activity. In contrast, during long-term incubations (24 h) with red blood cells PA-LOX induced significant (50%) hemolysis whereas rabbit ALOX15 was more or less ineffective. These data indicate the principle capability of PA-LOX of oxygenating membrane bound phospholipids which is likely to alter the barrier function of the biomembranes. Although the membrane oxygenase activity was lower than the fatty acid oxygenase activity of PA-LOX red blood cell membrane oxygenation might be of biological relevance for P. aeruginosa septicemia.

Mutagenesis of triad determinants of rat Alox15 alters the specificity of fatty acid and phospholipid oxygenation.

Among lipoxygenases ALOX15 orthologs are somewhat peculiar because of their capability of oxygenating polyenoic fatty acids even if they are incorporated in complex lipid-protein assemblies. ALOX15 orthologs of different species have been characterized before, but little is known about the corresponding rat enzyme. Since rats are frequently employed as models in biomedical research we expressed rat Alox15 as recombinant protein in pro- and eukaryotic expression systems and characterized the enzyme with respect to its enzymatic properties. The enzyme oxygenated free arachidonic acid mainly to 12S-HpETE with 15S-HpETE only contributing 10% to the product mixture. Multiple directed mutagenesis studies indicated applicability of the triad concept with particular importance of Leu353 and Ile593 as specificity determinants. Ala404Gly exchange induced subtle alterations in enantioselectivity suggesting partial applicability of the Coffa/Brash concept. Wildtype rat Alox15 and its 15-lipoxygenating Leu353Phe mutant are capable of oxygenating ester lipids of biomembranes and high-density lipoproteins. For the wildtype enzyme 13S-HODE and 12S-HETE were identified as major oxygenation products but for the Leu353Phe mutant 13S-HODE and 15S-HETE prevailed. These data indicate for the first time that mutagenesis of triad determinants modifies the reaction specificity of ALOX15 orthologs with free fatty acids and complex ester lipids in a similar way.

Inhibition of 12/15 lipoxygenase by curcumin and an extract from Curcuma longa L.

Curcumin (diferuloylmethane) is an orange-yellow secondary metabolic compound from the rhizome of turmeric (Curcuma longa L.), a spice often found in curry powder. It is one of the major curcuminoids of turmeric. For centuries, curcumin has been used in some medicinal preparations or as a food colouring agent. A variety of enzymes that are closely associated with inflammation and cancer were found to be modulated by curcumin. This paper summarized the results of the inhibitory effect of curcumin and a Curcuma longa L. ethanolic extract on lipoxygenase from the rat lung cytosolic fraction. The positional specificity determination of arachidonic acid dioxygenation by RP- and SP-HPLC methods showed that in a purified enzyme preparation from the rat lung cytosol the specific form of lipoxygenase (LOX) is present exhibiting 12/15-LOX dual specificity (with predominant 15-LOX activity). The inhibitory activity of curcumin and Curcuma longa extract on LOX from cytosolic fraction of rat lung was expressed in the percentage of inhibition and as IC50. Lineweaver-Burk plot analysis has indicated that curcumin is the competitive inhibitor of 12/15 LOX from the rat lung cytosolic fraction.

New pharmaceutical insights related to the pathways of PUFAs.

The fatty acyl structure represents the major lipid building block of practically all lipids and therefore is one of the most fundamental categories of these molecules. Fatty acids (FAs) differ particularly in their chain length, number of double bonds and position of the bonds in the chain. The number of double bonds in the unsaturated molecule of FA distinguishes monounsaturated FAs (MUFAs) and polyunsaturated FAs (PUFAs). In the living cell PUFAs represent the dominant substrates for the formation of biologically active compounds--octadecanoids, eicosanoids and docosanoids--classified as oxylipins or as PUFAnoids. The present review focuses only on the groups of PUFAnoids which biological activities comprise a "positive effect" for the cell. This group of omega-3 PUFAnoids consists of lipoxins, resolvins and protectins. All these biologically active lipids are formed mainly in the LOX-pathway. They are part of the cell mechanisms that contribute to the removal of inflammatory cells and restoration of tissue integrity. A new approach to an optimal anti-inflammatory model shows orientation to the dual COX/LOX-inhibition and the stimulation of the protective eicosanoids and docosanoids formation and its considerable therapeutic potential in managing of molecular mechanisms of chronic inflammatory processes.

[Identification of lipoxygenase expressing omega-6 dioxygenase activity by means of liquid chromatography]. / Identifikácia lipoxygenázy s omega-6 dioxygenázovou aktivitou kvapalinovou chromatografiou.

Lipoxygenases (LOX) represent a family of lipid peroxidising enzymes which catalyse the reaction of achiral polyunsaturated fatty acids by oxygen forming chiral peroxide products possessing high positional stereospecific purity. The four double bonds of arachidonic acid, the main substrate of animal LOX, present the position for a wide range of enzymatic modifications enabling eicosanoid creation, unique molecules with biological significance. In this study, lipoxygenase from rat lung cytoplasma was isolated and purified to 40-fold by combining hydrophobic and gel chromatography. The forming positional specific fatty acid hydroxyl-isomers were separated on a nonpolar system (RP-HPLC) and identified on a polar adsorbent (SP-HPLC). In the purified enzyme, dual positional specificity was demonstrated by the production of 12- and 15-HETE in the ratio of 1,0:1,38, which responds to the product spectrum of mammalian 15-LOX-1.