The N‐terminal β‐barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies

Christian, May; Höhne, Michaela; Gnau, Petra; Schwennesen, Karsten; Kindl, Helmut

doi:10.1046/j.1432-1327.2000.01105.x

Cited by 43 publications

(30 citation statements)

References 32 publications

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“…It therefore seems likely that different structural domains can fulfi ll this function in LOXs. While other studies reported that the ␤ -barrel domain mediates the binding to membranes ( 45,47 ), this domain could not contribute to a remarkable binding to liposomes in CspLOX1 under the tested conditions. It should be noted that the three tryptophan residues, which have been discussed to be involved in selective binding to PC in human 5-LOX ( 47 ), are not conserved in the ␤ -barrel domain of CspLOX1.…”

Section: The ␣ -Helical Extension At the N-terminus Facilitates Bindicontrasting

confidence: 69%

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

Newie¹,

Andreou²,

Neumann

et al. 2016

Journal of Lipid Research

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This article is available online at http://www.jlr.org PUFA oxygenation is a process that leads to the formation of bioactive lipid compounds with a diversity of biological functions in plants and animals ( 1, 2 ). Oxidation of PUFAs may be catalyzed by two major classes of enzymes, cyclooxygenases or ␣ -dioxygenases and lipoxygenases (LOXs) ( 3 ). Of the two, LOXs are nonheme iron-containing dioxygenases that are widely found in higher plants and animals, but have also been detected in some corals, mosses, fungi, and a number of bacteria ( 4, 5 ). Members of the LOX family catalyze the regio-and stereospecifi c oxygenation of PUFAs with one or more (1 Z ,4 Z )-pentadiene moieties leading to the formation of hydroperoxy PUFAs ( 6 ). LOX hydroperoxide products are precursors of important signaling compounds such as aldehydes and jasmonates in plants and leukotrienes, resolvins, and lipoxins in mammals ( 3 ). These signaling molecules play an important role in wound and defense responses as well as in aspects of plant development ( 7 ), while in mammals they function in infl ammation, asthma, and the development of atherosclerosis and cancer ( 1 ). Other than in higher organisms, very little is still known regarding the overall function of LOX products in prokaryotes and fungi ( 4, 5 ).As the regio-and stereospecifi city of the LOX reaction has an infl uence on the biological function of the product, many studies have focused on the molecular basis of this specifi city. In the case of arachidonic acid [20:4(n-6)], which is a typical mammalian LOX substrate, several Abstract In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The fi rst step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. Here we characterized CspLOX1, a phylogenetically distant LOX family member from Cyanothece sp. PCC 8801 and determined its crystal structure. In addition to the classical two domains found in plant, animal, and coral LOXs, we identifi ed an N-terminal helical extension, reminiscent of the long ␣ -helical insertion in Pseudomonas aeruginosa LOX. In liposome fl otation studies, this helical extension, rather than the ␤ -barrel domain, was crucial for a membrane binding function. Additionally, CspLOX1 could oxygenate 1,2-diarachidonyl-sn -glycero-3-phosphocholine, suggesting that the enzyme may act directly on membranes and that fatty acids bind to the active site in a tail-fi rst orientation. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9 R -and 11 R -hydroperoxides, respectively. Together these results reveal unifying structural features of LOXs and their function. While the core of the active site is important for lipoxygenation and thus highly conserved, peripheral domains functioning in membrane and substrate binding are more variable.

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Section: The ␣ -Helical Extension At the N-terminus Facilitates Bindicontrasting

confidence: 69%

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

Newie¹,

Andreou²,

Neumann

et al. 2016

Journal of Lipid Research

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“…As indicated above, other LOX isoforms, such as the human 5-LOX (13,20), the cucumber lipid body LOX (38), and the soybean 15-LOX-1 (10,39), are also capable of binding to biomembranes. For the 5-LOX (20) and the cucumber (38) linoleate 13-LOX, it has been suggested that their N-terminal ␤-barrel domains are responsible for the membrane binding activities.…”

Section: Table IV Catalytic Activities Of 15-lox Speciesmentioning

confidence: 93%

The N-terminal Domain of the Reticulocyte-type 15-Lipoxygenase Is Not Essential for Enzymatic Activity but Contains Determinants for Membrane Binding

Walther¹,

Anton²,

Wiedmann³

et al. 2002

Journal of Biological Chemistry

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The rabbit reticulocyte-type 15-lipoxygenase is capable of oxygenating biomembranes and lipoproteins without the preceding action of ester lipid cleaving enzymes. This reaction requires an efficient membrane binding, and the N-terminal ␤-barrel domain of the enzyme has been implicated in this process. To obtain detailed information on the structural requirements for membrane oxygenation, we expressed the rabbit wildtype 15-lipoxygenase, its ␤-barrel deletion mutant (catalytic domain), and several lipoxygenase point mutations as His-tagged fusion proteins in Escherichia coli and tested their membrane binding characteristics. We found that: (i) the ␤-barrel deletion mutant was catalytically active and its enzymatic properties (K M , V max , pH optimum, substrate specificity) were similar to those of the wild-type enzyme; (ii) when compared with the wildtype lipoxygenase, the membrane binding properties of the N-terminal truncation mutant were impaired but not abolished, suggesting a role of the catalytic domain in membrane binding; and (iii) Phe-70 and Leu-71 (constituents of the ␤-barrel domain) but also Trp-181, which is located in the catalytic domain, were identified as sequence determinants for membrane binding. Mutation of these amino acids to more polar residues (F70H, L71K, W181E) impaired the membrane binding capacity of the recombinant enzyme. These data indicate that the C-terminal catalytic domain of the rabbit 15-lipoxygenase is enzymatically active and that the membrane binding properties of the enzyme are determined by a concerted action of the N-terminal ␤-barrel and the C-terminal catalytic domain.

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“…Liposome suspensions corresponding to 1 µmol of phosphatidylcholine (50 µL) were incubated for 30 min at 25°C with LOX-1 or mini-LOX (2 µM each, corresponding to 100 pmol/test), alone or in the presence of 10 µM CaCl 2 or 10 µM CaCl 2 + 100 µM EDTA. The enzyme-liposome complexes were separated by flotation on a linear sucrose density gradient (34); then an enzymelinked immunosorbent assay (ELISA) was performed by coating the plate with enzyme-liposome complexes (50 µL/ well). Specific anti-LOX-1 monoclonal antibodies (35), diluted 1:400, were used as first antibody, and goat antimouse alkaline phosphatase conjugate (Bio-Rad, Richmond, CA), diluted 1:2000, as second antibody (35).…”

Section: Methodsmentioning

confidence: 99%

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

et al. 2001

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Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of R-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio-and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...

show abstract

The N‐terminal β‐barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies

Cited by 43 publications

References 32 publications

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

The N-terminal Domain of the Reticulocyte-type 15-Lipoxygenase Is Not Essential for Enzymatic Activity but Contains Determinants for Membrane Binding

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

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