Lipoxygenases

by Colin D. Funk, Ph.D., Alan R. Brash, Ph.D. and Kirk M. Maxey, M.D.

Lipoxygenases are a class of dioxygenase enzymes containing a catalytic non-heme, non-sulfur iron atom. Lipoxygenases catalyze the stereospecific insertion of molecular oxygen into polyunsaturated fatty acids containing an unconjugated (Z,Z)-1,4-pentadiene moiety. The products formed are optically active (S)- or (R)-hydroperoxides. Lipoxygenases are widely distributed throughout the plant and animal kingdoms.^1,2 Although they have not been found in bacteria or yeast, lipoxygenase gene sequences have been isolated from primitive species including alga, slime molds, and corals.

Most of our understanding about the lipoxygenase enzymes is derived by inference from the study of 15(S)-lipoxygenase isoforms from soybean (Glycine max). The first complete cDNA sequence, pure crystalline enzyme and three-dimensional structure, and mechanistic studies came from the soybean enzyme.^3-5 All mammalian lipoxygenases isolated so far have also proven to be (S)-lipoxygenases. They selectively add a single oxygen molecule to the pro-S face of the diallyl radical formed by hydrogen atom abstraction from the unsaturated fatty acid substrate. Lipoxygenases with (R) specificity are known from lower animal phyla including the 12(R)-lipoxygenase from echinoderms.⁶ The first purification, partial sequencing and cloning of an (R)-lipoxygenase from the gorgonian P. homomalla was completed last year.⁷ Although (R)-lipoxygenases have not yet been isolated from higher animals, it is certainly possible that the well documented (R)-HETE metabolites observed in tissues like cornea and skin could arise from this type of enzyme.

Early studies of mammalian lipoxygenases focused primarily on the enzymes found in different blood cell types, and categorized them into three main groups according to their positional specificity with respect to arachidonic acid.⁸ The three groups are 5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase from neutrophils, platelets, and reticulocytes, respectively. It has recently become clear that this classification oversimplifies the diversity of the lipoxygenase lineage. In the mouse, cDNAs for a 12(S)-lipoxygenase unique to epidermal cells and a mixed 12(S)/15(S)-lipoxygenase from macrophages have been recently discovered. The macrophage enzyme is very similar to the reticulocyte 15-lipoxygenase in amino acid sequence, gene structure, and oxygenation specificity, but has been classified as a leukocyte-type 12-lipoxygenase. Cloning experiments have also revealed an 8(S)-lipoxygenase in the mouse¹⁷, and a second, non-reticulocyte 15-lipoxygenase from human skin.⁹ As additional novel mammalian lipoxygenases are discovered, the older system of organization becomes ambiguous. A more comprehensive classification scheme based on sequence homology is presented in Figure 1.

The functions of lipoxygenase products in plants and animals are numerous and in some cases are still not well defined. In plants, the initial hydroperoxides undergo a variety of lyase, cleavage, and rearrangement reactions to form short-chain alcohols, vinyl ethers, aldehydes, and oxo acids.¹⁰ The most studied of these is Jasmonic acid, a cyclopentanoic acid derived from the 13-hydroperoxide of linoleate. Jasmonic acid has been shown to induce growth inhibition, senescence and gene expression leading to production of defensive proteins such as proteinase-inhibitors. The volatile methyl ester of jasmonic acid also exhibits these effects when applied in the vapor phase, providing a mechanism for remote signaling from wounded plants to other nearby individuals.

In mammals, the 5-lipoxygenase pathway has been the major focus of study due to the pronounced pro-inflammatory role of the leukotrienes. The leukotrienes were well known to medicine as the slow reacting substance of anaphylaxis (SRS-A) long before their chemical structure was elucidated.^11,12 The recent approvals of 5-lipoxygenase inhibitors for the clinical treatment of asthma are clear evidence for the importance of lipoxygenase metabolites in human biology.

Although less well characterized, the 12-lipoxygenase pathway in humans may also have an important role in the progression of dysplasia and metastasis of some carcinomas.¹³ The 12-lipoxygenase activity of various tumor cell lines has been correlated with their metastatic potential. This may be due to the activity of 12(S)-HETE on both the adhesiveness and motility of tumor cells, as well as its similar effects on adjacent endothelial cells.

The 15-lipoxygenase pathway has been considered a clinical target off and on for several years. This is due to the suspicious association of oxidized fatty acids with the fatty placques that characterize atherosclerosis. The 13-hydroperoxide of linoleate is a prominent oxidized lipid in these lesions, implicating a lipoxygenase in their pathogenesis.^14-16 However, it has not been clear which lipoxygenase from what cell type might be involved, or whether non-enzymatic free radical oxygenation was playing a dominant role. The massive amounts of 15-lipoxygenase enzyme present in the human reticulocytes clearly imply some biological role, leaving much to be discovered in this promising field in the future.

References

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17. Brash, A. R., et al. J. Biol. Chem. In press.

Additional Figures

Positional Selectivity of Lipoxygenases

Lipoxygenases have historically been described according to their positional selectivity in the oxygenation of arachidonic acid, the one common in vitro substrate to all known lipoxygenases. But just how defining a characteristic is regional specificity anyway? This convention only seems to apply nicely to the human platelet 12-lipoxygenase, which is an arachidonate-specific lipoxygenase with positional selectivity for carbon 12. EPA is the only other substrate fatty acid of interest to this enzyme, which is essentially inactive toward linoleic acid. In contrast, the porcine leukocyte 12-lipoxygenase utilizes linoleate at twice the rate of arachidonic acid. The porcine enzyme seems to be better described as a linoleate 13-lipoxygenase with a reduced but significant affinity for 20-carbon fatty acids as alternative substrates. Of course, the classical 13-lipoxygenase is the soybean 15-lipoxygenase, so named because of its active oxygenation C-15 in arachidonate, a substrate that it is never exposed to in its natural environment within the soybean plant. It should be remembered that the soybean 15-lipoxygenase incorporates oxygen into C-13 of linoleate at 3 times the rate of the corresponding reaction with arachidonic acid. Thus, substrate specificity links the soybean enzyme to the porcine leukocyte enzyme, despite the fact that they oxygenate arachidonate on different carbons.

Work by David Sloane has shown just how fickle a property the regional specificity of lipoxygenase enzymes can be. Working with the recombinant mammalian 15-lipoxygenase, he prepared site-directed mutants which demonstrate this fact.^1,2 The native enzyme sequence places the residues isoleucine and methionine at positions 417 and 418, respectively. Mutating both of these to the smaller amino acid valine resulted in an active lipoxygenase in which the substrate could slip deeper into the binding pocket, placing C-10 closer to the iron atom than C-13. The lipoxygenase product of the mutant was 12(S)-HETE. Since changing 2 amino acid residues changed this enzyme from a 15-lipoxygenase into a 12-lipoxygenase, regioselectivity may not be informative about evolutionary relationships. Perhaps the novel lipoxygenases can be incorporated into a new classification based on overall sequence homology.

References

1. Sloane, D.L., et al. Nature 354, 149-152 (1991).
2. Gan, Q., et al. J. Biol. Chem. 271, 25412-25418 (1996).

Gene Linked to Severe Asthma Identified by New Assay

Platelet Activating Factor (PAF) was identified and characterized in 1980 as a phospholipid mediator released by sensitized rabbit basophils.¹ The ability of this new mediator to aggregate platelets at 1 × 10^-10 M stimulated numerous studies which demonstrated widespread, potent inflammatory activity.² Fifteen years later, a novel extracellular phospholipase A₂ enzyme associated with the LDL fraction of human plasma was purified and cloned.³ Identified as PAF acetylhydrolase (PAF-AH), this 45 kD protein appeared to act as the endogenous inactivator of PAF. Potential clinical importance was quickly recognized as both PAF and other pro-inflammatory, oxidized phospholipids could be held in check by this enzyme. A recombinant form of PAF-AH is currently in Phase II clinical trials for indications such as ARDS.

Genetic analysis soon identified a mutation in the gene encoding PAF-AH which led to a deficiency of plasma PAF-AH activity.⁴ This gene is remarkably common in Japanese populations, with the heterozygote frequency estimated as high as 27%. Analysis of children with asthma showed that persons with the defective gene were overrepresented in the group of childhood asthmatics presenting with the most severe symptoms. It has now been clearly demonstrated that the possession of a single defective copy of the PAF-AH gene is a prominent risk factor for the development of severe asthma, as well as for the development of atherosclerotic coronary artery disease.

Along with the increase in clinical interest in PAF-AH comes a need for an accurate, sensitive test for PAF-AH activity. Activity tests may be of equal or greater importance than immunometric sandwich-type assays for PAF-AH, since much of the PAF-AH enzyme which circulates in the blood may no longer be catalytically active. Such a test could be used to screen populations at risk (such as children presenting with asthma) and identify those most susceptible to life threatening attacks. A simple, colorimetric assay for PAF-AH activity has recently been introduced by Cayman Chemical (Catalog Number 760901). The new assay represents a significant improvement on the older method based on PAF radiolabeled with ¹⁴C-acetate. The use of 2-thio PAF as substrate eliminates the poor recoveries and large variances typical of the radiometric assay. PAF-AH readily accepts the 2-thio PAF analog as a substrate and converts it to the free thiol, 2-thio lyso-PAF. This product is detected using Ellman's reagent according to the Scheme I.

References

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3. Tjoelker, L.W. et al. Nature 374, 549 (1995)
4. Stafforini, D.M. et al. J. Clin. Invest. 97, 2784 (1996).