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

Abstract

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.

Keywords: Eicosanoids; Enzymology; Evolution; Leukotrienes; Lipid metabolism; Lipoxygenase.

Fig. 1. Catalytic activities of ALOX15 orthologs.

A) The lipoxygenase reaction consists of 4 elementary reactions (hydrogen abstraction, radical rearrangement, dioxygen insertion, peroxy radical reduction). To initiate the reaction the ferrous LOX is first activated by peroxide-dependent oxidation to a ferric form [modified from (Ivanov et al., 2010)]. B) The lipohydroperoxidase activity is initiated when a lipid hydroperoxide (ROOH) is bound at the active site of the enzyme. The enzyme then catalyzes a homolytic cleavage of the hydroperoxy bond, which leads to the formation of an oxygen-centered alkoxy radical, a hydroxyl and oxidizes the ferrous iron to a ferric form. Then the enzyme binds a linoleic acid molecule (or an alterative reductant such as guaiacol) and releases a carbon-centered linoleic radical. This reaction reduces the ferric LOX back to its ferrous form to start the next catalytic cycle. The released radical intermediates may then initiate free radical secondary reactions leading to the formation of mixed oxygenated and non-oxygenated linoleic acid dimers. C) The leukotriene synthase activity of various LOX-isoforms involved a homolytic cleavage of the hydroperoxy group and a hydrogen abstracton from a bisallylic methylene. These consecutive reaction steps lead to the formation of a fatty acid biradical, which stabilizes by epoxide formation.

Fig. 2. Kinetic progress curve of arachidonic oxygenation by pure rabbit ALOX15.

When peroxide-free fatty acids are used as substrates the kinetic progress curve of the ALOX15 reaction can be separated in three periods. i) Kinetic lag-phase: The oxygenation reaction starts with a kinetic lag-phase, in which product formation increases with time. ii) Linear phase: The lag phase is followed by a more or less linear part of the progress curve, in which the reaction rate does not change. iii) Suicidal inactivation phase: During the final part of the progress curve the reaction rate decreases with time, which has been related to suicidal inactivation of the enzyme.

Fig. 3. Molecular docking studies of a phospholipid molecule at the active site of rabbit ALOX15.

To construct these images the following sets of X-ray coordinates (PDB entries) were employed: rabbit ALOX15 (2POM), soybean LOX-1 (1YGE), and phospholipid (4G32). The GOLD program with default parameters was used for docking the phospholipid into the active sites of rabbit ALOX15 conformers and soybean LOX1. For preparation of images the Accelrys Discovery Studio 4.0 Visualizer was employed. The amino acids labeled represent examples for steric clashes with the phospholipid substrate. A) rabbit ALOX15 (non-liganded conformer), B) rabbit ALOX15 (liganded conformer, C) soybean LOX1. The docking studies were carried out by Kumar Reddy Kakularam from the Department of Animal Sciences, School of Life Science, University of Hyderabad (India) and the National Institute of Animal Biotechnology, Hyderabad (India). Permission for publication was granted.

Fig. 4. Reaction specificity of rabbit ALOX15 with arachidonic acid isomers.

The different arachidonic acid isomers are aligned at the active site of rabbit ALOX15 in such a way that different bisallylic methylenes are located in close proximity to the enzyme bound non-heme iron so that hydrogen abstraction from these carbon atoms is possible. For instance, for the 4,7,10,13-isomer (ω−7) hydrogen is abstracted only from C12 and oxygen is inserted only at C14 (n+2 radical rearrangement). With this substrate rabbit ALOX15 exhibits a singular positional specificity as indicated by product analysis (GC/MS). With arachidonc acid (5,8,11,14-isomer, ω−6) the iron is located between the bisallylic methylenes C13 and C10 (but closer to C13) and thus, hydrogen can be abstracted from both carbon atoms with strong preference of the C13 bisallylic methylene. Oxygen is then preferentially (85%) inserted at C15 but to a lesser extent (15%) also at C12 (n+2 radical rearrangement in both cases). With this substrate the enzyme exhibits a dual positional specificity. For the 6,9,12,15-isomer (ω−5) an even more pronounced dual positional specificity was observed since oxygen was inserted in similar quantities at C17 and C14.

Fig. 5. Triad concept of positional specificity of ALOX15 orthologs.

For ALOX15 orthologs arachidonic acid slides into the substrate-binding pocket with its methyl end ahead and is bound at the active site by hydrophobic interactions and probably by π-π-interactions of the substrates double bonds with aromatic active site amino acids. The amino acids, which align with Phe353, Ile418 and Ile593 of the rabbit enzyme, form the bottom of the substrate binding pocket and the methyl terminus of the fatty acid substrate interacts with the side chains of these amino acids. For the 15-lipoxygenating rabbit ALOX15 these positions are occupied by bulky residues (Phe353, Ile418, Ile593) so that the substrate fatty acid does not penetrate as deep into the substrate-binding pocket (left side of the images). Thus, the bisallylic methylene C13 of the arachidonic acid is bound in close proximity to the catalytic non-heme iron and this alignment results in major 15-lipoxygenation. In 12-lipoxygenating ALOX15 orthologs (mouse, rats, pigs, cattle) either of these positions is occupied by a less space-filling amino acid, which allows the substrate fatty acid to penetrate deeper into the active site (right side of the image) and the black arrows indicate the direction of substrate movement. This movement approaches the bisallylic methylene C10 of arachidonc acid to the non-heme iron so that hydrogen abstraction from C10 becomes possible. In the 12-lipoxygenating panel (right side of the image) the amino acid exchanges are indicated, which lead to alterations in the reaction specificity during in vitro mutagenesis.

Fig. 6. Presence of 4-fold C repeats in the 3’-UTR of murine ALOX15 mRNA.

The murine alox15 mRNAs do not contain the repetitive DICE element, which has been implicated in translational regulation of the rabbit and human ALOX15 mRNA. Instead on the genomic level various 4-fold C-repeats (CCCC or TCCC) are present and these sequences may functionally substitute for the lacking DICE element.

Fig. 7. Structural properties of rabbit ALOX15.

(A) Iron ligand sphere of rabbit ALOX15. Four histidines (His361, His366, His541, His545), the N-terminal Ile/663 and a water molecule are the 1^st order iron ligands of rabbit ALOX15 (B). Overlay of the two structures (ligand-free conformer, ligand-bound conformer) of the rabbit ALOX15. Ligand-free conformer A is indicated in grey and ligand-bound structure (conformer B) in yellow. The non-heme iron is also shown. It can be seen that helix 2 is strongly dislocated upon ligand binding by about 12 Å. Rotation of the active site helix 18 can also be seen. (C) Crystal structure of rabbit ALOX15 dimers. In the crystals rabbit ALOX15 forms heterodimers consisting of a ligand-free (conformer A) and a ligand-bound (conformer B) monomer. Inset: The residues contributing to the interaction between the two monomers are indicated and a number of leucine and tryptophane residues contribute. The program VMD 1.4.8 version (University of Illinois) and the coordinates of rabbit LOX complex (PDB code: 2P0M) were used to create these images.

Fig. 8:. Ligand induced oligomerization of rabbit ALOX15 and impact of interdomain interface mutants on enzyme oligomerization.

Low-resolution models of rabbit ALOX15 were calculated on the bases of small angle X-ray scattering data in the absence (upper panel) and presence (lower panel) of a 10-fold molar excess of 13S-HODE as active site ligand. Structural models of ALOX15 dimers were generated by rigid body refinement applying P2 symmetry on conformers A keeping the intermonomer interface as shown in the crystal structure (PDB code: 2P0M). For the tetramer model of the Leu183Glu+Leu192Glu mutants in complex with 13(S)-HODE P222 symmetry was considered. The four catalytic domains are shown in gray, N-terminal domains in red and the α2 helixes in orange. The tetrameric structure shows significant difference to the dimers with P2 symmetry. Images were modified according to (Ivanov et al., 2012).

Fig. 9. Biological relevance of ALOX15.

(A) Principle mechanisms, by which ALOX15 orthologs exhibit their bioactivity. (B) Physiological and patho-physiological processes, in which ALOX15 orthologs have been implicated.

Fig. 10. Alox15-deficient mice suffer from more severe experimental autoimmune encephalitis when compared with alox15 sufficient controls.

Experimental autoimmune encephalitis (EAE) was induced in 8–10 week old female alox15-deficient mice (LOX-KO; n=7) and corresponding wildtype controls (WT; n=7) by subcutaneous immunization with 200 µg MOG_35–55 peptide (purity >95%, Pepceuticals, Leicester, UK) emulsified in an equal volume of PBS and complete Freund’s adjuvant containing 6 mg/ml Mycobacterium tuberculosis H37Ra (Difco, FranklinLakes, NJ). Bordetella pertussis toxin (200 ng, PTX, List Biological Laboratories, Campbell, CA) was administered intraperitoneally at day 0 and 2 post-immunization. Mice were weighed and scored daily as follows: 0 = no disease; 1 = complete tail paralysis; 2 = abnormal gait, hindlimb paresis; 3 = hindlimb plegia; 4 = paraplegia and forelimb weakness; 5 = moribund or death due to EAE. Mann-Whitney-U-test *=p<0.05. The experiments were carried out by Silvina Romero-Suarez and Carmen Infante-Duarte at the Institute for Medical Immunology, Charité Universitätsmedizin Berlin. Data were kindly provided and publication was allowed by the authors.