Publications

• Functional characterization of genetic enzyme variations in human lipoxygenases.

  Horn T., Reddy Kakularam K., Anton M., Richter C., Reddanna P., Kuhn H.

  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 homeos ... >> More

  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<0.1%. This data suggest that there appears to be an evolutionary pressure on the coding regions of the lipoxygenase genes preventing the accumulation of loss-of-function variations in the human population. << Less

  Redox Biol. 1:566-577(2013) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Mutations of Triad Determinants Changes the Substrate Alignment at the Catalytic Center of Human ALOX5.

  Ivanov I., Golovanov A.B., Ferretti C., Canyelles-Nino M., Heydeck D., Stehling S., Lluch J.M., Gonzalez-Lafont A., Kuehn H.

  For the specificity of ALOX15 orthologs of different mammals, the geometry of the amino acids Phe353, Ile418, Met419, and Ile593 ("triad determinants") is important, and mutagenesis of these residues altered the reaction specificity of these enzymes. Here we expressed wild-type human ALOX5 and its ... >> More

  For the specificity of ALOX15 orthologs of different mammals, the geometry of the amino acids Phe353, Ile418, Met419, and Ile593 ("triad determinants") is important, and mutagenesis of these residues altered the reaction specificity of these enzymes. Here we expressed wild-type human ALOX5 and its F359W/A424I/N425M/A603I mutant in Sf9 insect cells and characterized the catalytic differences of the two enzyme variants. We found that wild-type ALOX5 converted arachidonic acid mainly to 5(<i>S</i>)-hydroperoxyeicosatetraenoic acid (HpETE). In contrast, 15(<i>S</i>)- and 8(<i>S</i>)-H(p)ETE were formed by the mutant enzyme. In addition to arachidonic acid, wild-type ALOX5 accepted eicosapentaenoic acid (EPA) as substrate, but C18 fatty acids were not oxygenated. The quadruple mutant also accepted linoleic acid and α- and γ-linolenic acid as substrate. Structural analysis of the oxygenation products and kinetic studies with stereospecifically labeled 11(<i>S</i>)- and 11(<i>R</i>)-deutero-linoleic acid suggested alternative ways of substrate orientation at the active site. <i>In silico</i> docking studies, molecular dynamics simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations confirmed this hypothesis. These data indicate that "triad determinant" mutagenesis alters the catalytic properties of ALOX5 abolishing its leukotriene synthase activity but improving its biosynthetic capacity for pro-resolving lipoxins. << Less

  ACS Chem. Biol. 14:2768-2782(2019) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Oxidative inactivation of human 5-lipoxygenase in phosphatidylcholine vesicles.

  De Carolis E., Denis D., Riendeau D.

  Human 5-lipoxygenase is a non-heme iron protein which possesses 5-oxygenase, leukotriene A4 synthase and pseudoperoxidase activities and which undergoes a rapid irreversible inactivation during these reactions. The inactivation of the enzyme was dependent on the structural characteristics of the s ... >> More

  Human 5-lipoxygenase is a non-heme iron protein which possesses 5-oxygenase, leukotriene A4 synthase and pseudoperoxidase activities and which undergoes a rapid irreversible inactivation during these reactions. The inactivation of the enzyme was dependent on the structural characteristics of the substrate for the reaction, on O2 concentration and on exposure to phospholipids and calcium. The apparent first-order rate constant for enzyme inactivation (k(in)) was 0.6 min(-1) during the oxygenation of arachidonic acid in air-saturated buffer containing phosphatidylcholine vesicles and Ca2+. The rate of enzyme inactivation was dependent on the substrate for the reaction and was about threefold slower during the oxygenation of 5,8-icosadienoic acid and 12(S)-hydroxyicosatetraenoic acid compared with arachidonic acid. Lowering the 02 concentration to 60 microM during the oxygenation of arachidonic acid also caused a 2.5-fold decrease in k(in) without affecting the initial rate of the reaction resulting in an increase in both 5-hydroperoxyicosatetraenoic acid (5-HPETE) and leukotriene A4 accumulation. The concentration of 02 for half-maximal activity (initial rate and product accumulation) was approximately 10 microM. In contrast, the activity and the rate of inactivation during the leukotriene A4 synthase reaction with exogenous 5-HPETE (k(in)=2.0 min(-1) were independent of 02 concentration. A rapid inactivation of the enzyme was also observed during aerobic incubation with phosphatidylcholine vesicles and Ca2+ in the absence of substrate, with a sequential loss of the oxygenase (t1/2 = 0.5 min) and pseudoperoxidase (t1/2 = 7 min) activities. Protection against this turnover-independent inactivation was observed in the presence of the selective reversible 5-lipoxygenase inhibitor L-739,010 ([1S, 5R] 3-cyano-1-(3-furyl)-6-(6-[3-(3 alpha-hydroxy-6,8-dioxyabicyclo [3.2.11 octanyl)] pyridin-2-ylmethoxy) naphthalene) and by prior treatment of vesicles with sodium borohydride and, to a lesser extent, by glutathione peroxidase. The results show that the inactivation of 5-lipoxygenase in phospholipid vesicles is dependent on the structure of the unsaturated fatty acid substrate for the reaction, on the concentration of oxygen and on a turnover-independent oxidation at the active-site leading to the sequential loss of the oxygenase and pseudoperoxidase activities of the enzyme. << Less

  Eur. J. Biochem. 235:416-423(1996) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• The suppression of 5-lipoxygenation of arachidonic acid in human polymorphonuclear leucocytes by the 15-lipoxygenase product (15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid: structure-activity relationship and mechanism of action.

  Petrich K., Ludwig P., Kuehn H., Schewe T.

  (15S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) suppresses in ionophore-A23187-stimulated human polymorphonuclear leucocytes (PMN) the conversion of exogenous arachidonic acid into leukotriene B(4) (LTB4) and (5S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). However, contr ... >> More

  (15S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) suppresses in ionophore-A23187-stimulated human polymorphonuclear leucocytes (PMN) the conversion of exogenous arachidonic acid into leukotriene B(4) (LTB4) and (5S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). However, contrary to earlier suggestions, 15-HETE is not a genuine 5-lipoxygenase inhibitor under these conditions, but rather suppresses the 5-lipoxygenation of arachidonic acid by switching-over of substrate utilization, as judged from a sizeable formation of labelled (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetr aen oic acid (5,15-diHETE) from 15-[1(-14)C]HETE. Identical results were obtained with human recombinant 5-lipoxygenase. In PMN the formation of 5,15-diHETE is strongly stimulated by either hydroperoxypolyenoic fatty acids or arachidonic acid, suggesting a crucial role of the hydroperoxide tone of the cell. A comparison of a selection of hydroxypolyenoic fatty acids with respect to their capability of suppressing 5-lipoxygenation of arachidonic acid revealed that 15-mono-hydroxyeicosanoids throughout exhibit the highest inhibitory potencies, whereas the other HETEs, 5,15-diHETE as well as octadecanoids, are modest or poor inhibitors. The R and S enantiomers of 15-HETE do not differ from each other, excluding a receptor-like binding of the 15-hydroxy group. << Less

  Biochem. J. 314:911-916(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities.

  Rouzer C.A., Matsumoto T., Samuelsson B.

  The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chro ... >> More

  The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chromatography, and HPLC on anion-exchange and hydroxyapatite columns demonstrated that LTA4 synthase activity copurified with the 5-lipoxygenase with similar recoveries and increases in specific activity. Furthermore, the two enzymatic activities coeluted exactly on three different HPLC systems. Maximal activity of purified LTA4 synthase required the addition of three nondialyzable stimulatory factors, two of which were cytosolic and one of which was membrane-bound. These findings were identical for 5-lipoxygenase activity. When incubated with arachidonic acid, the purified 5-lipoxygenase converted approximately equal to 15% of its endogenously generated 5-hydroperoxyicosatetraenoic acid (5-HPETE) to LTA4. LTA4 production was more efficient when the enzyme utilized 5-HPETE generated from arachidonic acid than when 5-HPETE was exogenously supplied as substrate. These findings suggest that a single protein from human leukocytes possesses 5-lipoxygenase and LTA4 synthase activities and that the synthesis of LTA4 from 5-HPETE is controlled by the same complex multicomponent system that regulates the 5-lipoxygenase reaction. << Less

  Proc Natl Acad Sci U S A 83:857-861(1986) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

  Smyrniotis C.J., Barbour S.R., Xia Z., Hixon M.S., Holman T.R.

  5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of ... >> More

  5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation. << Less

  Biochemistry 53:4407-4419(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.