The lipoxygenases are related to each other based upon their similar genetic structure and dioxygenation activity. However, one lipoxygenase, ALOXE3, while having a lipoxygenase genetic structure, possesses relatively little dioxygenation activity; rather its primary activity appears to be as an isomerase that catalyzes the conversion of hydroperoxy unsaturated fatty acids to their 1,5-epoxide,hydroxyl derivatives.
Lipoxygenases are found in eukaryotes (plants, fungi, animals, protists); while the third domain of terrestrial life, thearchaea, possesses proteins with a slight (~20%) amino acid sequence similarity to lipoxygenases, these proteins lack iron-binding residues and therefore are not projected to possess lipoxygenase activity.[2]
Based on detailed analyses of 15-lipoxygenase 1 and stabilized 5-lipoxygenase, lipoxygenase structures consist of a 15kilodalton N-terminalbeta barrel domain, a small (e.g. ~0.6 kilodalton) linker inter-domain (seeProtein domain § Domains and protein flexibility), and a relatively large C-terminal catalytic domain which contains the non-heme iron critical for the enzymes' catalytic activity.[3] Most of the lipoxygenases (exception, ALOXE3) catalyze the reactionPolyunsaturated fatty acid + O2 → fatty acidhydroperoxide in four steps:
the rate-limiting step of hydrogen abstraction from a bisallylicmethylene carbon to form a fatty acid radical at that carbon
rearrangement of the radical to another carbon center
addition of molecular oxygen (O2) to the rearranged carbon radical center thereby forming a peroxy radical(—OO·) bond to that carbon
reduction of the peroxy radical to its corresponding anion (—OO−)
The (—OO−) residue may then be protonated to form a hydroperoxide group (—OOH) and further metabolized by the lipoxygenase to e.g.leukotrienes,hepoxilins, and variousspecialized pro-resolving mediators, or reduced by ubiquitous cellular glutathioneperoxidases to a hydroxy group thereby forming hydroxylated (—OH) polyunsaturated fatty acids such as thehydroxyeicosatetraenoic acids andHODEs (i.e. hydroxyoctadecaenoic acids).[3]
Lipoxygenases depend on the availability of their polyunsaturated fatty acid substrates which, particularly in mammalian cells, is normally maintained at extremely low levels. In general, variousphospholipase A2s and diacylglycerol lipases are activated during cell stimulation, proceed to release these fatty acids from their storage sites, and thereby are key regulators in the formation of lipoxygenase-dependent metabolites.[3] In addition, cells, when so activated, may transfer their released polyunsaturated fatty acids to adjacent or nearby cells which then metabolize them through their lipoxygenase pathways in a process termed transcellular metabolism or transcellular biosynthesis.[6]
These enzymes are most common in plants where they may be involved in a number of diverse aspects of plant physiology including growth and development, pest resistance, and senescence or responses to wounding.[7] In mammals a number of lipoxygenasesisozymes are involved in the metabolism ofeicosanoids (such asprostaglandins,leukotrienes andnonclassic eicosanoids).[8] Sequence data is available for the following lipoxygenases:
An illustrative transformation involving a hydroperoxide lyase. Here cis-3-hexenal is generated fromlinolenic acid to the hydroperoxide by the action of a lipoxygenase followed by the lyase.[10]
With the exception of the gene encoding 5-LOX (ALOX5), which is located on chromosome 10q11.2, all six humanLOX genes are located on chromosome 17.p13 and code for a single chain protein of 75–81kilodaltons that consists of 662–711 amino acids. MammalianLOX genes contain 14 (ALOX5,ALOX12,ALOX15,ALOX15B) or 15 (ALOX12B,ALOXE3)exons with exon/intron boundaries at highly conserved positions.[11][12] The 6 human lipoxygenases along with some of the major products that they make, as well as some of their associations with genetic diseases, are as follows:[11][13][14][15][16]
Arachidonate 15-lipoxygenase-1 (ALOX15) (EC1.13.11.33;InterPro: IPR001885), also termed 15-lipoxygenase-1, erythrocyte type 15-lipoxygenase (or 15-lipoxygenase, erythrocyte type), reticulocyte type 15-lipoxygenase (or 15-lipoxygenase, reticulocyte type), 15-LO-1, and 15-LOX-1. It metabolizes arachidonic acid principally to1) 15-hydroperoxyeiocatetraenoic acid (15-HpETE) which is further metabolized to15-hydroxyicosatetraenoic acid (15-HETE) but also to far smaller amounts of2) 12-hydroperoxyeicosatetraenoic acid (12-HpETE) which is further metabolized to12-hydroxyeicosatetraenoic acid and possibly thehepoxilins. ALOX15 actually preferslinoleic acid over arachidonic acid, metabolizing linoleic acid to 12-hydroperoxyoctadecaenoic acid (13-HpODE) which is further metabolized to13-hydroxyoctadecadienoic acid (13-HODE). ALOX15 can metabolize polyunsaturated fatty acids that are esterified tophospholipids and/or to thecholesterol, i.e.cholesterol esters, inlipoproteins. This property along with its dual specificity in metabolizing arachidonic acid to 12-HpETE and 15-HpETE are similar to those of mouse Alox15 and has led to both enzymes being termed 12/15-lipoxygenases.
Arachidonate 15-lipoxygenase type II (ALOX15B), also termed 15-lipoxygenase-2, 15-LOX-2, and 15-LOX-2.[17] It metabolizes arachidonic acid to 15-hydroperoxyeicosatetraenoic (15-HpETE) which is further metabolized to15-hydroxyicosatetraenoic acid. ALOX15B has little or no ability to metabolize arachidonic acid to 12-hydroperoxeiocosatetraenoic acid (12-(HpETE) and only minimal ability to metabolize linoleic acid to 13-hydroperoxyoctadecaenoic acid (13-HpODE).
Arachidonate 12-lipoxygenase, 12R type (ALOX12B), also termed 12R-lipoxygenase, 12R-LOX, and 12R-LO.[18] It metabolizes arachidonic acid to 12R-hydroxyeicosatetraenoic acid but does so only with low catalytic activity; its most physiologically important substrate is thought to be asphingosine which contains a very long chain (16-34 carbons) omega-hydroxyl fatty acid that is in amide linkage to thesn-2 nitrogen of sphingosine at itscarboxy end and esterified to linoleic acid at its omega hydroxyl end. In skin epidermal cells, ALOX12B metabolizes the linoleate in this esterified omega-hydroxyacyl-sphingosine (EOS) to its 9R-hydroperoxy analog. Inactivating mutations of ALOX12B are associated with the human skin disease, autosomal recessivecongenital ichthyosiform erythroderma (ARCI).[18][19]
Epidermis-type lipoxygenase (ALOXE3), also termed eLOX3 and lipoxygenase, epidermis type.[20] Unlike other lipoxygenases, ALOXE3 exhibits only a latent dioxygenase activity. Rather, its primary activity is as a hydroperoxide isomerase that metabolizes certain unsaturated hydroperoxy fatty acids to their corresponding epoxy alcohol and epoxy keto derivatives and thereby is also classified as ahepoxilin synthase. While it can metabolize 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE) to theRstereoisomers of hepoxilins A3 and B3, ALOXE3 favors metabolizingR hydroperoxy unsaturated fatty acids and efficiently converts the 9(R)-hydroperoxy analog of EOS made by ALOX15B to its 9R(10R),13R-trans-epoxy-11E,13R and 9-keto-10E,12Z EOS analogs.[19] ALOXE3 is thought to act with ALOX12B in skin epidermis to form the latter two EOS analogs; inactivation mutations of ALOX3 are, similar to inactivating mutations in ALOX12B, associated with autosomal recessivecongenital ichthyosiform erythroderma in humans.[19][20] Inactivating mutations in ALOX3 are also associated with the human disease lamellar ichthyosis (seeIchthyosis § Types – item 5 in the table).
Two lipoxygenases may act in series to make di-hydroxy or tri-hydroxy products that have activities quite different than either lipoxyenases' products. This serial metabolism may occur in different cell types that express only one of the two lipoxygenases in a process termed transcellular metabolism. For example, ALOX5 and ALOX15 or, alternatively, ALOX5 and ALOX12 can act serially to metabolize arachidonic acid intolipoxins (see 15-Hydroxyeicosatetraenoic acid §§ Further metabolism andActivities of 15(S)-HpETE, 15(S)-HETE, 15(R)-HpETE, 15(R)-HETE, and 15-oxo-ETE andLipoxin § Synthesis) while ALOX15 and possibly ALOX15B can act with ALOX5 to metabolizeeicosapentaenoic acid to resolvin D's (seeResolvin § Biochemistry and production).
The mouse is a common model to examine lipoxygenase function. However, there are some key differences between the lipoxygenases between mice and men that make extrapolations from mice studies to humans difficult. In contrast to the 6 functional lipoxygenases in humans, mice have 7 functional lipoxygenases and some of the latter have different metabolic activities than their humanorthologs.[11][19][21] In particular, mouse Alox15, unlike human ALOX15, metabolizes arachidonic acid mainly to 12-HpETE and mouse Alox15b, in contrast to human ALOX15b, is primarily an 8-lipoxygenase, metabolizing arachdionic acid to 8-HpETE; there is no comparable 8-HpETE-forming lipoxygenase in humans.[22]
Alox5 appears to be similar in function to human ALOX5.
Alox12 differs from human ALOX12, which preferentially metabolizes arachidonic acid to 12-HpETE but also to substantial amounts of 15-HpETE, in that metabolizes arachidonic acid almost exclusively to 12-HpETE.
Alox15 (also termed leukocyte-type 12-Lox, 12-Lox-l, and 12/15-Lox) differs from human ALOX15, which under standard assay conditions metabolizes arachidonic acid to 15-HpETE and 12-HpETE products in an 89 to 11 ratio, metabolizes arachidonic acid to 15-HpETE and 12-HpETE in a 1 to 6 ratio, i.e. its principal metabolite is 12-HpETE. Also, human ALOX15 prefers linoleic acid over arachidonic acid as a substrate, metabolizing it to 13-HpODE while Alox15 has little or no activity on linoleic acid. Alox15 can metabolize polyunsaturated fatty acids that are esterified tophospholipids andcholesterol (i.e.cholesterol esters). This property along with its dual specificity in metabolizing arachidonic acid to 12-HpETE and 15-HpETE are similar to those of human ALOX15 and has led to both enzymes being termed 12/15-lipoxygenases.
Alox15b (also termed 8-lipoxygenase, 8-lox, and 15-lipoxygenase type II), in contrast to ALOX15B which metabolizes arachidonic acid principally to 15-HpETE and to a lesser extent linoleic acid to 13-HpODE, metabolizes arachidonic acid principally to 8S-HpETE and linoleic acid to 9-HpODE. Alox15b is as effective as ALOX5 in metabolizing 5-HpETE to leukotrienes.
Alox12e (12-Lox-e, epidermal-type 12-Lox) is an ortholog to the human ALOX12P gene which has suffered damaging mutations and is not expressed. ALox12e prefers methyl esters over non-esterified polyunsaturated fatty acid substrates, metabolizing linoleic acid ester to its 13-hydroperoxy counterpart and to a lesser extent arachidonic acid ester to its 12-hydroperoxy counterpart.
Alox12b (e-LOX2, epidermis-type Lox-12) appears to act similarly to ALOX12B to metabolize the linoleic acid moiety of EOS to its 9R-hydroperoxy counterpart and thereby contribute to skin integrity and water impermeability; mice depleted to Alox12b develop a severe skin defect similar to Congenital ichthyosiform erythroderma. Unlike human ALOX12B which cam metabolize arachidonic acid to 12R-HETE at a low rate, Alox12b does not metabolize arachidonic acid as free acid but dose metabolize arachidonic acid methyl ester to its 12R-hydroperoxy counterpart.
Aloxe3 (epidermis-type Lox-3, eLox3) appears to act similarly to ALOXe3 in metabolizing the 9R-hydoperoxy-linoleate derivative of EOS to its epoxy and keto derivatives and to be involved in maintaining skin integrity and water impermeability. AloxE3 deletion leads to a defect similar to congenital ichthyosiform erythroderma.
Rabbit 15-lipoxygenase (blue) with inhibitor (yellow) bound in the active site
There are several lipoxygenase structures known including: soybean lipoxygenase L1 and L3, coral 8-lipoxygenase, human 5-lipoxygenase, rabbit 15-lipoxygenase and porcine leukocyte 12-lipoxygenase catalytic domain. The protein consists of a small N-terminalPLAT domain and a major C-terminal catalytic domain (seePfam database), which contains theactive site. In both plant and mammalian enzymes, the N-terminal domain contains an eight-stranded antiparallel β-barrel, but in the soybean lipoxygenases this domain is significantly larger than in the rabbit enzyme. The plant lipoxygenases can be enzymatically cleaved into two fragments which stay tightly associated while the enzyme remains active; separation of the two domains leads to loss of catalytic activity. The C-terminal (catalytic) domain consists of 18-22 helices and one (in rabbit enzyme) or two (in soybean enzymes) antiparallel β-sheets at the opposite end from the N-terminal β-barrel.
The iron atom in lipoxygenases is bound by four ligands, three of which are histidine residues.[23] Six histidines are conserved in all lipoxygenase sequences, five of them are found clustered in a stretch of 40 amino acids. This region contains two of the three zinc-ligands; the other histidines have been shown[24] to be important for the activity of lipoxygenases.
The two long central helices cross at the active site; both helices include internal stretches ofπ-helix that provide threehistidine (His) ligands to the active site iron. Two cavities in the major domain of soybean lipoxygenase-1 (cavities I and II) extend from the surface to the active site. The funnel-shaped cavity I may function as a dioxygen channel; the long narrow cavity II is presumably a substrate pocket. The more compact mammalian enzyme contains only one boot-shaped cavity (cavity II). In soybean lipoxygenase-3 there is a third cavity which runs from the iron site to the interface of theβ-barrel and catalytic domains. Cavity III, the iron site and cavity II form a continuous passage throughout the protein molecule.
The active site iron is coordinated by Nε of three conserved His residues and one oxygen of the C-terminal carboxyl group. In addition, in soybean enzymes theside chain oxygen ofasparagine is weakly associated with the iron. In rabbit lipoxygenase, this Asn residue is replaced with His which coordinates the iron via Nδ atom. Thus, the coordination number of iron is either five or six, with a hydroxyl or water ligand to a hexacoordinate iron.
Details about the active site feature of lipoxygenase were revealed in the structure of porcine leukocyte 12-lipoxygenase catalytic domain complex[23][25] In the 3D structure, the substrate analog inhibitor occupied a U-shaped channel open adjacent to the iron site. This channel could accommodate arachidonic acid without much computation, defining the substrate binding details for the lipoxygenase reaction. In addition, a plausible access channel, which intercepts the substrate binding channel and extended to the protein surface could be counted for the oxygen path.
Soybean Lipoxygenase 1 exhibits the largest H/Dkinetic isotope effect (KIE) on kcat (kH/kD) (81 near room temperature) so far reported for a biological system. Recently, an extremely elevated KIE of 540 to 730 was found in a double mutant Soybean Lipoxygenase 1.[26] Because of the large magnitude of the KIE, Soybean Lipoxygenase 1 has served as the prototype for enzyme-catalyzed hydrogen-tunneling reactions.
Human proteins expressed from the lipoxygenase family includeALOX12,ALOX12B,ALOX15,ALOX15B,ALOX5, andALOXE3. While humans also possess theALOX12P2 gene, which is anortholog of the well-expressedAlox12P gene in mice, the human gene is apseudogene; consequently, ALOX12P2 protein is not detected in humans.[27]
^Choi J, Chon JK, Kim S, Shin W (February 2008). "Conformational flexibility in mammalian 15S-lipoxygenase: Reinterpretation of the crystallographic data".Proteins.70 (3):1023–32.doi:10.1002/prot.21590.PMID17847087.S2CID40013415.
^Capra V, Rovati GE, Mangano P, Buccellati C, Murphy RC, Sala A (2015). "Transcellular biosynthesis of eicosanoid lipid mediators".Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids.1851 (4):377–82.doi:10.1016/j.bbalip.2014.09.002.PMID25218301.
^Vick BA, Zimmerman DC (1987). "Oxidative Systems for Modification of Fatty Acids: The Lipoxygenase Pathway".Oxidative systems for the modification of fatty acids: The Lipoxygenase Pathway. Vol. 9. pp. 53–90.doi:10.1016/b978-0-12-675409-4.50009-5.ISBN9780126754094.
^abcKrieg, P; Fürstenberger, G (2014). "The role of lipoxygenases in epidermis".Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids.1841 (3):390–400.doi:10.1016/j.bbalip.2013.08.005.PMID23954555.
^Haeggström, J. Z.; Funk, C. D. (2011). "Lipoxygenase and leukotriene pathways: Biochemistry, biology, and roles in disease".Chemical Reviews.111 (10):5866–98.doi:10.1021/cr200246d.PMID21936577.
^Romano M, Cianci E, Simiele F, Recchiuti A (2015). "Lipoxins and aspirin-triggered lipoxins in resolution of inflammation".European Journal of Pharmacology.760:49–63.doi:10.1016/j.ejphar.2015.03.083.PMID25895638.
^Steczko J, Donoho GP, Clemens JC, Dixon JE, Axelrod B (1992). "Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement".Biochemistry.31 (16):4053–4057.doi:10.1021/bi00131a022.PMID1567851.
