Dagmar Heydeck

Molecular enzymology of lipoxygenases.

Lipoxygenases (LOXs) are lipid peroxidizing enzymes, implicated in the pathogenesis of inflammatory and hyperproliferative diseases, which represent potential targets for pharmacological intervention. Although soybean LOX1 was discovered more than 60years ago, the structural biology of these enzymes was not studied until the mid 1990s. In 1993 the first crystal structure for a plant LOX was solved and following this protein biochemistry and molecular enzymology became major fields in LOX research. This review focuses on recent developments in molecular enzymology of LOXs and summarizes our current understanding of the structural basis of LOX catalysis. Various hypotheses explaining the reaction specificity of different isoforms are critically reviewed and their pros and cons briefly discussed. Moreover, we summarize the current knowledge of LOX evolution by profiling the existence of LOX-related genomic sequences in the three kingdoms of life. Such sequences are found in eukaryotes and bacteria but not in archaea. Although the biological role of LOXs in lower organisms is far from clear, sequence data suggests that this enzyme family might have evolved shortly after the appearance of atmospheric oxygen on earth.

In vivo action of 15-lipoxygenase in early stages of human atherogenesis.

Oxidative modification of low density lipoprotein has been suggested as patho-physiologically relevant process in atherogenesis and the lipid peroxidizing enzyme 15-lipoxygenase may be involved. For experimental evidence on the in vivo action of this enzyme in the time course of plaque formation we analyzed the lipid extracts of lesional areas representing various stages of human atherogenesis for the occurrence of specific 15-lipoxygenase products. In advanced human lesions the degree of oxygenation of the lesion lipids measured as hydroxy linoleic acid/linoleic acid ratio varied between 0.2 and 3.2%. Here an unspecific pattern of oxygenated lipids that did not differ from the pattern formed during copper-catalyzed LDL oxidation was detected. In both cases an enantiomer ratio (S/R-ratio) of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) of approximately 1:1 was found. In young human lesions which were obtained from the collection of the pathological determinants of atherosclerosis in youth (PDAY) program the hydroxy linoleic acid/linoleic acid ratio was much smaller (variation between 0.05 and 0.6%), and a significant share of specific 15-lipoxygenase products was detected (S/R-ratio of 13-hydroxy linoleic acid of 54 +/- 3.1/46 +/- 3.1 [mean +/- SD]). These data suggest that the 15-lipoxygenase is enzymatically active on endogenous substrates in young human lesions and thus, may be of patho-physiological importance for early atherogenesis. In advanced human plaques the 15-lipoxygenase may be functionally silent and specific lipoxygenase products formed in earlier stages may be decomposed or superimposed by large amounts of nonenzymatic lipid peroxidation products.

Redox Control in Mammalian Embryo Development

The development of an embryo constitutes a complex choreography of regulatory events that underlies precise temporal and spatial control. Throughout this process the embryo encounters ever changing environments, which challenge its metabolism. Oxygen is required for embryogenesis but it also poses a potential hazard via formation of reactive oxygen and reactive nitrogen species (ROS/RNS). These metabolites are capable of modifying macromolecules (lipids, proteins, nucleic acids) and altering their biological functions. On one hand, such modifications may have deleterious consequences and must be counteracted by antioxidant defense systems. On the other hand, ROS/RNS function as essential signal transducers regulating the cellular phenotype. In this context the combined maternal/embryonic redox homeostasis is of major importance and dysregulations in the equilibrium of pro- and antioxidative processes retard embryo development, leading to organ malformation and embryo lethality. Silencing the in vivo expression of pro- and antioxidative enzymes provided deeper insights into the role of the embryonic redox equilibrium. Moreover, novel mechanisms linking the cellular redox homeostasis to gene expression regulation have recently been discovered (oxygen sensing DNA demethylases and protein phosphatases, redox-sensitive microRNAs and transcription factors, moonlighting enzymes of the cellular redox homeostasis) and their contribution to embryo development is critically reviewed.

Regulation of cellular 15-lipoxygenase activity on pretranslational, translational, and posttranslational levels

In mammalian cells, enzymatic lipid peroxidation catalyzed by 12/15-lipoxygenases is regulated by pretranslational, translational, and posttranslational processes. In rabbits, rats, and mice induction of experimental anemia leads to a systemic up-regulation of 12/15-lipoxygenases expression. In addition, interleukins-4 and -13 were identified as strong up-regulators of this enzyme in human and murine monocyte/macrophages and in the lung carcinoma cell line A549, and the interleukin-4(13) cell surface receptor as well as the signal transducer and activator of transcription 6 (STATG) appears to be involved in the signal transduction cascade. On the level of translation, 15-lipoxygenase synthesis is blocked by the binding of regulatory proteins to a characteristic gurnine-cytosine-rich repetitive element in the 3′-untranslated region of the rabbit 15-lipoxygenase mRNA, and the formation of such 15-lipoxygenase mRNA/protein complexes was identified as molecular reason for the translational inactivity of the 15-lipoxygenase mRNA in immature red blood cells. However, proteolytic breakdown of the regulatory proteins which were recently identified as hnRNP K and hnRNP E1 overcomes translational inhibition during later stages of reticulocyte maturation. For maximal intracellular activity, 12/15-lipoxygenases require a rise in cytosolic calcium concentration inducing a translocation of the enzyme from the cytosol to cellular membranes as well as small amounts of preformed hydroperoxides which act as essential activators of the enzymes. 12/15-Lipoxygenases undergo irreversible suicide inactivation during fatty acid oxygenation, and this process may be considered an element of down-regulation of enzyme activity. Suicide inactivation and proteolytic breakdown may contribute to the disappearance of functional 12/15-lipoxygenase at later stages of erythropoiesis.

Structural and functional biology of arachidonic acid 15-lipoxygenase-1 (ALOX15)

Lipoxygenases (LOX) form a family of lipid peroxidizing enzymes, which have been implicated in a number of physiological processes and in the pathogenesis of inflammatory, hyperproliferative and neurodegenerative diseases. They occur in two of the three domains of terrestrial life (bacteria, eucarya) and the human genome involves six functional LOX genes, which encode for six different LOX isoforms. One of these isoforms is ALOX15, which has first been described in rabbits in 1974 as enzyme capable of oxidizing membrane phospholipids during the maturational breakdown of mitochondria in immature red blood cells. During the following decades ALOX15 has extensively been characterized and its biological functions have been studied in a number of cellular in vitro systems as well as in various whole animal disease models. This review is aimed at summarizing the current knowledge on the protein-chemical, molecular biological and enzymatic properties of ALOX15 in various species (human, mouse, rabbit, rat) as well as its implication in cellular physiology and in the pathogenesis of various diseases.

Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling

Abstract 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.

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.

Leukotriene signaling in the extinct human subspecies Homo denisovan and Homo neanderthalensis. Structural and functional comparison with Homo sapiens

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.

Development of myeloproliferative disease in 12/15-lipoxygenase deficiency.

To the editor: We read with interest the report by Kinder and colleagues at the Wistar Institute[1][1] along with their related studies[2][2],[3][3] showing that 12/15-lipoxygenase (LOX)–deficiency in (C57BL/6, N11) mice leads to severe myeloproliferative disease (MPD). Moderate splenomegaly with

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

Significance Lipoxygenases are lipid-peroxidizing enzymes that have been classified according to their reaction specificity. ALOX15 (12/15-lipoxygenase) has been implicated in inflammatory resolution via biosynthesis of antiinflammatory and proresolving lipoxins. We found that lower mammals including lower primates express arachidonic acid 12-lipoxygenating ALOX15 orthologs, whereas higher primates express 15-lipoxygenating enzymes. Gibbons constitute the missing link interconnecting 12- and 15-lipoxygenating ALOX15 orthologs. To explore the evolutionary driving force for this specificity alteration, we quantified the lipoxin synthase activity of 12- and 15-lipoxygenating ALOX15 orthologs and observed that the lipoxin synthase activities of 15-lipoxygenating enzymes were significantly higher. These results suggest an evolution of ALOX15 specificity, which optimizes the biosynthetic capacity for 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, ratPhe353Ala) 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). 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.