Lipoxygenase

Lipoxygenases (LOX) are a family of (non heme) iron containing enzymes, more specifically oxidative enzymes, most of which catalyze the dioxygenation of polyunsaturated fatty acids in lipids containing a cis,cis 1,4 pentadiene into cell signaling agents that serve diverse roles as autocrine signals that regulate the function of their parent cells, paracrine signals that regulate the function of nearby cells, and endocrine signals that regulate the function of distant cells. 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, the archaea, 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.

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. In mammals a number of lipoxygenases isozymes are involved in the metabolism of eicosanoids (such as prostaglandins, leukotrienes and nonclassic eicosanoids). Sequence data is available for the following lipoxygenases:

Plants express a variety of cytosolic lipoxygenases (; ) as well as what seems to be a chloroplast isozyme. Plant lipoxygenase in conjunction with hydroperoxide lyases are responsible for many fragrances and other signalling compounds. One example is cis 3 hexenal, the odor of freshly cut grass.

With the exception of the gene encoding 5 LOX (ALOX5), which is located on chromosome 10q11.2, all six human LOX genes are located on chromosome 17. p13 and code for a single chain protein of 75–81 kilodaltons that consists of 662–711 amino acids. Mammalian LOX genes contain 14 (ALOX5, ALOX12, ALOX15, ALOX15B) or 15 (ALOX12B, ALOXE3) exons with exon/intron boundaries at highly conserved positions. 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:

Arachidonate 5 lipoxygenase (ALOX5) (; ), also termed 5 lipoxygenase, 5 LOX, and 5 LO. Major products: it metabolizes arachidonic acid to 5 hydroperoxy eicostetraeoic acid (5 HpETE) which is converted to 1) 5 hydroxyicosatetraenoic acid (5 HETE) and then to 5 oxo eicosatetraenoic acid (5 oxo ETE), 2) leukotriene A4 (LTA4) which may then be converted to leukotriene B4 (LTB4) or leukotriene C4 (LTC4) (LTC4 may be further metabolized to leukotriene D4 [LTD4] and then to leukotriene E4 [LTE4]), or 3) acting in series with ALOX15, to the specialized pro resolving mediators, lipoxins A4 and B4. ALOX5 also metabolizes eicosapentaenoic acid to a set of metabolites that contain 5 double bounds (i. e. 5 HEPE, 5 oxo EPE, LTB5, LTC5, LTD5, and LTE5) as opposed to the 4 double bond containing arachidonic acid metabolites. The enzyme, when acting in series with other lipoxygenase, cyclooxygenase, or cytochrome P450 enzymes, contributes to the metabolism of eicosapentaenoic acid to E series resolvins (see ) and of docosahexaenoic acid to D series resolvins (see ). These resolvins are also classified as specialized pro resolving mediators. Arachidonate 12 lipoxygenase (ALOX12) (; ), also termed 12 lipoxygenase, platelet type platelet lipoxygenase (or 12 lipoxygenase, platelet type) 12 LOX, and 12 LO. It metabolizes arachidonic acid to 12 hydroperoxyeiocsatetraeoic acid (12 HpETE) which is further metabolized to 12 hydroxyeicosatetraenoic acid (12 HETE) or to various hepoxilins (also see 12 hydroxyeicosatetraenoic acid). Arachidonate 15 lipoxygenase 1 (ALOX15) (; ), 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 to 1) 15 hydroperoxyeiocatetraenoic acid (15 HpETE) which is further metabolized to 15 hydroxyicosatetraenoic acid (15 HETE) but also to far smaller amounts of 2) 12 hydroperoxyeicosatetraenoic acid (12 HpETE) which is further metabolized to 12 hydroxyeicosatetraenoic acid and possibly the hepoxilins. ALOX15 actually prefers linoleic acid over arachidonic acid, metabolizing linoleic acid to 12 hydroperoxyoctadecaenoic acid (13 HpODE) which is further metabolized to 13 hydroxyoctadecadienoic acid (13 HODE). ALOX15 can metabolize polyunsaturated fatty acids that are esterified to phospholipids and/or to the cholesterol, i. e. cholesterol esters, in lipoproteins. 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. It metabolizes arachidonic acid to 15 hydroperoxyeicosatetraenoic (15 HpETE) which is further metabolized to 15 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. 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 a sphingosine which contains a very long chain (16 34 carbons) omega hydroxyl fatty acid that is in amide linkage to the sn 2 nitrogen of sphingosine at its carboxy 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 recessive congenital ichthyosiform erythroderma (ARCI). Epidermis type lipoxygenase (ALOXE3), also termed eLOX3 and lipoxygenase, epidermis type. 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 a hepoxilin synthase. While it can metabolize 12S hydroperoxyeicosatetraenoic acid (12S HpETE) to the R stereoisomers of hepoxilins A3 and B3, ALOXE3 favors metabolizing R 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. 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. 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 to phospholipids and cholesterol (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.

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 terminal PLAT domain and a major C terminal catalytic domain (see Pfam database), which contains the active 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. 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 to be important for the activity of lipoxygenases. The two long central helices cross at the active site; both helices include internal stretches of π helix that provide three histidine (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 the β 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 the side chain oxygen of asparagine 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 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.