**Inhibition of Soybean Lipoxygenases – Structural and Activity Models for the Lipoxygenase Isoenzymes Family**

Veronica Sanda Chedea1 and Mitsuo Jisaka2

*1Laboratory of Animal Biology, National Research Development Institute for Animal Biology and Nutrition (IBNA), 2Faculty of Life Sciences and Biotechnology, Shimane University, 1Romania 2Japan* 

### **1. Introduction**

108 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

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Lipoxygenases (EC 1.13.11.12, linoleate:oxygen, oxidoreductases, LOXs) which are widely found in plants, fungi, and animals, are a large monomeric protein family with non-heme, non-sulphur, iron cofactor containing dioxygenases that catalyze the oxidation of polyunsaturated fatty acids (PUFA) as substrate with at least one 1*Z*, 4*Z*-pentadiene moiety such as linoleic, linolenic and arachidonic acid to yield hydroperoxides (Gardner, 1991).

Fig. 1. Lipoxygenase substrates, linoleic, α- linolenic and arachidonic acid.

Theorell et al. (1947) succeeded in crystallizing and characterizing lipoxygenase (LOX) from soybeans and since then among plant LOXs, soybean lipoxygenase isozyme 1 (LOX-1) can be regarded as the mechanistic paradigm for these nonheme iron dioxygenases (Coffa et al., 2005; Minor et al., 1996; Fiorucci et al., 2008).

Designing agents to modulate activities of the variety of so closely homologous enzymes, such as different LOXs, require an intimate knowledge of their 3D structures, as well as information about metabolism of the potential xeno- or endobiotics. So far only the structures of soybean isozymes LOX-1 and LOX-3 have been determined for native enzymes, and several structures of their and rabbit 15-LOX (from reticulocytes) molecular complexes with inhibitors are known. Due to lack of sufficiently purified human enzymes most of the structural research has been done on soybean LOX (Skrzypczak-Jankun et al., 2003).

Inhibition of Soybean Lipoxygenases – Structural

into 9-LOXs, 13-LOXs, or 9/13-LOXs.

hydroperoxyoctadecatrienoic acid.

**4.1 In plants** 

**4.2 In humans** 

**4. Biological and metabolic functions** 

development (Feussner & Wasternack, 2002).

and Activity Models for the Lipoxygenase Isoenzymes Family 111

formed by the 1*Z*,4*Z*-pentadiene unit. In most LOX reactions, particularly those in plants, the resulting hydroperoxy groups are in *S*-configuration, while one mammalian LOX and some marine invertebrate LOXs produce *R*-hydroperoxides. Even in the reactions of such "*R*-LOXs", the antarafacial rule of hydrogen removal and oxygen addition is conserved. In cases of plant LOXs, including soybean LOXs, the usual substrates are C18 polyunsaturated fatty acids (linoleic and -linolenic acids), and the products are their 9*S*- or 13*S*-hydroperoxides (Fig. 2B). Most plant LOXs react with either one of the regio-specificity, while some with both. Therefore, based on the regio-specificty, plant LOXs are classified

Fig. 2. LOX reaction showing the principal steps of LOX reaction (Panel A), and the actual

Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes of plants (Feussner & Wasternack, 2002). The hydroperoxy polyunsaturated fatty acids, synthesized by the action of various highly specialised forms of lipoxygenases, are substrates of at least seven different enzyme families (Feussner & Wasternack, 2002). Signaling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among the numerous products. Thus, the lipoxygenase pathway becomes an initial step in the interaction of plants with pathogens, insects, or abiotic stress and at distinct stages of

Besides polyunsaturated fatty acids, H2O2, fatty acid hydroperoxides, and synthetic organic hydroperoxides support the lipoxygenase-catalyzed xenobiotic oxidation the major reactions documented thus far including oxidation, epoxidation, hydroxylation, sulfoxidation, desulfuration, dearylation, and N-dealkylation (Kulkarni, 2001). It is noteworthy that lipoxygenases are also capable of glutathione conjugation of certain xenobiotics (Kulkarni, 2001). Available data suggest that lipoxygenases contribute to in vivo

metabolism of endobiotics and xenobiotics in mammals (Kulkarni, 2001).

reactions of plant LOXs and -linolenic acid (Panel B). HPOTE:

Understanding the mechanism of inhibition of LOXs can have profound effect in the development of many anti-cancer and anti-inflammatory drugs. On the basis of the available LOX data it was suggested that a combination of LOX modulators might be needed to shift the balance of LOX activities from procarcinogenic to anticancerogenic as a novel strategy for cancer chemoprevention (Skrzypczak-Jankun et al., 2003).

The aim of the present study is to present knowledge on different lipoxygenases having the soybean lipoxygenases as a structural and activity template for their inhibition by natural antioxidant compounds as theoretical approach for food biochemistry and medical applications.

#### **2. Lipoxygenase structure and activity**

The three-dimensional structure of soybean lipoxygenase-1 has been determined to 2.3 Å resolution by single crystal X-ray diffraction methods (Boyington et al., 1993). It is a twodomain, single-chain prolate ellipsoid of dimensions 90 x 65 x 60 Å with a molecular mass of 95 kDa. The 839 residues are organised in two domains: one 146 residue N-terminal domain (domain I), and a major, 693 residue C-terminal domain (domain II) (Prigge et al., 1997). Overall, the three-dimensional structure of lipoxygenase-1 shows a helical content of 38.0% and a β-sheet content of 13.9%. The structure of another crystal form of soybean lipoxygenase-1 determined to 1.4 Å resolution (Minor et al., 1996) showed very similar results. The structure of lipoxygenase-3, another soybean lipoxygenase isozyme (Skrzypczak-Jankun, 1997) shows that the lipoxygenase-3 isozyme is very similar in structure despite significant differences in sequence: 857 residues vs 839, deletions at 7 positions, insertions at 25 positions, and substitutions at 224 residues (72% identity).

Soybean seed isoenzymes are 94–97 kDa monomeric proteins with distinct isoelectric points ranging from about 5.7 to 6.4, and can be distinguished by optimum pH, substrate specificity, product formation and stability (Siedow, 1991; Mack et al., 1987). LOX-1 is the smallest in size (838 amino acids; 94 kDa), exhibits maximal activity at pH 9.0 and converts linoleic acid preferentially into the 13-hydroperoxide derivative. LOX-2 is characterized by a larger size (865 amino acids; 97 kDa), by a peak of activity at pH 6.8, and forms equal amounts of the 13-and 9-hydroperoxide compounds (Loiseau et al., 2001). LOX-2 oxygenates the esterified unsaturated fatty acid moieties in membranes in contrast to LOX-1 which only uses free fatty acids as substrates (Maccarrone et al., 1994). LOX-3 (857 amino acids; 96.5 kDa) exhibits its maximal activity over a broad pH range centred around pH 7.0 and displays a moderate preference for producing a 9-hydroperoxide product. It is the most active isoenzyme with respect to both carotenoid cooxidation and production of oxodienoic acids (Ramadoss, 1978).

#### **3. Lipoxygenase reaction**

The initial step of LOX reaction is removal of a hydrogen atom from a methylene unit between double bonds in substrate fatty acids (Fig. 2A). The resulting carbon radical is stabilized by electron delocalization through the double bonds. Then, a molecular oxygen is added to the carbon atom at +2 or –2 position from the original radical carbon, forming a peroxy radical as well as a conjugated *trans*,*cis*-diene chromophore. The peroxy radical is then hydrogenated to form a hydroperoxide. The initial hydrogen removal and the following oxygen addition occur in opposite (or antarafacial) sides related to the plane formed by the 1*Z*,4*Z*-pentadiene unit. In most LOX reactions, particularly those in plants, the resulting hydroperoxy groups are in *S*-configuration, while one mammalian LOX and some marine invertebrate LOXs produce *R*-hydroperoxides. Even in the reactions of such "*R*-LOXs", the antarafacial rule of hydrogen removal and oxygen addition is conserved.

In cases of plant LOXs, including soybean LOXs, the usual substrates are C18 polyunsaturated fatty acids (linoleic and -linolenic acids), and the products are their 9*S*- or 13*S*-hydroperoxides (Fig. 2B). Most plant LOXs react with either one of the regio-specificity, while some with both. Therefore, based on the regio-specificty, plant LOXs are classified into 9-LOXs, 13-LOXs, or 9/13-LOXs.

Fig. 2. LOX reaction showing the principal steps of LOX reaction (Panel A), and the actual reactions of plant LOXs and -linolenic acid (Panel B). HPOTE: hydroperoxyoctadecatrienoic acid.
