**4. Biological and metabolic functions**

### **4.1 In plants**

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

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

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

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

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

novel strategy for cancer chemoprevention (Skrzypczak-Jankun et al., 2003).

applications.

acids (Ramadoss, 1978).

**3. Lipoxygenase reaction** 

**2. Lipoxygenase structure and activity** 

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 development (Feussner & Wasternack, 2002).

#### **4.2 In humans**

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).

Inhibition of Soybean Lipoxygenases – Structural

action by LOX (Borrellia et al., 1999).

**5.2 LOX inhibition in cancer** 

Guthrie & Carroll, 1999).

(Pidgeon et al., 2007).

**5.3 Mechanisms of lipoxygenase inhibition** 

showing similar LOX activity, was found (Trono et al., 1999).

preferentially expressed during the development of various cancers.

(Szymanowska et al., 2009) by the action of this enzyme.

and Activity Models for the Lipoxygenase Isoenzymes Family 113

implicated in the generation of the flavour and aroma in many plant products, in the decolourisation of pigments and in the potential of compromising the anti-oxidant status (Casey, 1999). In pasta the involvement of LOX in colour loss is demonstrated by positive correlation between the decrease of β-carotene content after pastification and LOX activities in semolina. In addition to this, the hydroperoxidation and bleaching activities of LOX are highly correlated demonstrating that the bleaching might be ascribable to a co-oxidative

During pasta processing in which the maximal pigment degradation by LOX activity occurs (Borrellib et al., 1999), it is shown that externally added β-carotene can act as inhibitor of the LOX-catalysed linoleate hydroperoxidation and an inverse relation between the % of carotenoid loss and the initial carotenoid content in semolina from durum varieties,

The complete characterisation of lipoxygenase from pea seeds (*Pisum sativum var. Telephone L.*) gives possibility to avoid destructive influence during food processing and storage

Molecular studies of the well-known relationship between polyunsaturated fatty acid metabolism and carcinogenesis have revealed novel molecular targets for cancer chemoprevention and treatment (Lipkin et al., 1999; Willett, 1997; Klurfeld & Bull, 1997;

The role of lipoxygenase in the development and progression of cancer is complex due to the variety of lipoxygenase genes that have been identified in humans, in addition to different profiles of lipoxygenase observed between studies on human tumor biopsies and experimentally induced animal tumor models (Pidgeon et al., 2007). The literature emerging on the role of lipoxygenases in tumor growth, for the most part, suggests that distinct lipoxygenase isoforms, whose expression are lost during the progression of cancer, may exhibit anti-tumor activity, while other isoforms may exert pro-tumorigenic effects and are

The involvement of 5-lipoxygenase and 12-lipoxygenase in human cancer progression is now supported by a growing body of literature. The involvement of 15-lipoxygenase-1 in colorectal cancer involves its implication in carcinogenesis having pro-carcinogenic as well as anti-carcinogenic roles (Bhattacharya et al., 2009). The co-localization of these enzymes and the similarities of their bioactions on cancer cell growth suggest that the simultaneous inhibition of these enzymes may represent novel and promising therapeutic approaches in selected cancer types (Pidgeon et al., 2007). Therefore, when targeting the regulation of arachidonic acid metabolism, blocking 5-lipoxygenase, 12-lipoxygenase and 15 lipoxygenase-1 without altering the expression of the anti-carcinogenic 15-lipoxygenase-2 may be the most effective, however at present no drug recapitulates these capabilities

In general, lipoxygenase inhibitors can bind covalently to iron or form the molecular complexes blocking access to iron (Skrzypczak-Jankun et al., 2007). It was pointed out by Walther et al., that a course of inhibition, by the drug ebselen, (noncompetitive vs competitive) and its reversibility depend on the oxidation state of iron, i.e. whether the enzyme is catalytically silent with Fe2+ when it binds covalently, causing irreversible

Recent reviews describe the role of lipoxygenase in cancer (Bhattacharya et al., 2009; Pidgeon et al., 2007; Moreno, 2009), inflammation (Duroudier et al., 2009; Hersberger, 2010) and vascular biology (Chawengsub et al., 2009; Mochizuki & Kwon, 2008) and for an extensive presentation of the role of eicosanoids in prevention and management of diseases the reader is referred to the review of Szefel et al. (2011).
