Biochemistry Of Vitamin E

All 6-hydroxychromanols that constitute the vitamin E family are plant products of well-defined biosynthetic routes. All photosynthetic organisms synthesize the vitamin. Synthesis has not been documented in any other organisms, and plant products provide the only natural dietary sources. Early studies concluded that a-T is formed in both photosynthetic and nonphotosynthetic tissue of higher plants, concentrated in the chloroplasts (51-52). Other tocopherols and tocotrienols are in higher concentration in nonphotosynthetic tissues (53). In Calendula officinalis leaves, a-T was only present in chloroplasts, whereas /-and 5-T were found in the chloroplasts, mitochondria, and microsomes (54). No tocopherols were present in Golgi membranes and cytosol. Biosynthesis of the tocopherols occurred primarily in the chloroplasts (55-58). Most vitamin E partitions into the lipid phase of the choloroplast membrane with the phytyl side chain embedded within the membrane bilayer (56). Orientation of the vitamin E occurs through interaction of the benzoquinone ring with the carbonyls of triacylglycerol esters (56). Such localization and orientation have been established in mammalian cells as well.

1.3.1. Biosynthesis Formation of Homogentisic Acid. Synthesis of vitamin E by higher plants is quite well understood. Early studies that defined the synthesis were reviewed by Threlfall (52) and Draper (59). Later reviews include those by Hess (56) and Bramley et al. (60), which provide insight into studies that characterize the enzymological features of the biosynthesis. Studies mostly completed in the 1960s identified the shikimic acid pathway present in plants, algae, and bacteria but not in animals (59) as a key pathway yielding homogentisic acid. The pathway (Figure 1.6) proceeds through the p-hydroxyphenylpyruvic acid intermediate, forming homogentisic acid, which constitutes the p-benzoquinone ring of the chromanol structure.

FIGURE 1.6 Biosynthesis of homogentisic acid. (Modified from Refs. 52, 59.

Homogentisic acid provides the backbone structure for further formation of the tocopherols and plastoquinones (59). Also, p-hydroxyphenylpyruvic acid is converted through tyrosine to ubiquinone. Conversion to homogentisic acid is catalyzed by p-hyroxyphenylpyruvic acid dioxygenase (HPPDase, p-hydroxyphenylpyruvate: oxygen oxidoreductase, hydroxylating, decarboxylating, EC, EC The HPPDase inserts two oxygen molecules, oxidatively decarboxylates, and rearranges the side chain of p-hydroxyphenylpyruvic acid to form homogentisic acid (60-63). In mammals, HPPDase functions in the degradation of aromatic amino acids. The subcellular location, purification, and cloning of genes of HPPDase from carrot cells (63) preceded accomplishment of further research with Arabidopsis sp. mutants (62). Conversion of Homogentisic Acid to Tocopherols. Conversion of homogentisic acid to the tocopherols includes the following steps:

1. Polyprenyltransferase Reaction: Addition of the phytyl side chain results from the reaction of homogentisic acid with phytyl-diphosphate (pyrophosphate) (Figure 1.7). Polyprenyltransferase catalyzes with the simultaneous prenylation reaction, decarboxylation, and release of pyrophosphate to form 2-methyl-6-phytylbenzoquinol, which constitutes the intermediate for synthesis of the tocopherols (60, 64). The polyprenyltransferases catalyze condensation reactions of homogentisic acid with phytyl-diphosphate, geranylgeranyl-diphosphate, or solanesyldiphosphate to form tocopherols, tocotrienols, and plastoquinones, respectively (64). Phytyl-diphosphate also provides the isoprenoid tail for the synthesis of phylloquinones (vitamin K1) and the chlorophylls (52, 64-66). Collakova and DellaPenna (64) successfully cloned gene products from Synechocystis sp. PCC6803 and Arabidopsis sp. that encode polyprenyltransferases specific for tocopherol synthesis. Loci PDS1 and PDS2 that had previously been characterized when mutated, decreased the levels of tocopherols and plastoquinones in Arabidopsis sp. The PDS1 locus encodes for p-hydroxyphenylpyruvate dioxygenase. Locus PDS2 was proposed to be responsible for synthesis of a polyprenyltransferase that catalyzes the conversion of homogentisic acid to either 2-methyl-6-phytylbenzoquinol or 2-demethylplastoquinol-9 (62). The PDS1 and PDS2 mutants in Arabidopsis sp. were proved to be deficient in plastoquinone and tocopherols (62). The PDS2 mutation was thought to affect a step of the plastoquinone-tocopherol pathway after the HPPDase reaction, most likely the polyprenyltransferase reaction.

2. 2-Methyl-6-Phytylbenzoquinol Methyl Transferase Reaction: 2-Methyl-6-phytylbenzoquinol is methylated by a methyltransferase

FIGURE 1.7 Conversion of homogentisic acid to tocopherols by the action of polyprenyltransferase and tocopherol cyclase. (Modified from Refs. 60, 64.)

to form 2,3-dimethyl-6-phytylbenzoquinol. This compound is the immediate precursor of /-T (67). In 2002, Shintani et al. (67) identified a putative 2-methyl-6-phytylbenzoquinol methyltransferase gene (SLL0418) from the Synechocystic sp. PCC6803 genome that encodes the methyltransferase. The enzyme catalyzes methylation of C-3 of 2-methyl-6-solanylbenzoquinol in the terminal step of plastoquinone synthesis. The enzyme was described as playing a more important role in determining the tocopherol profile than in determining total tocopherol content (67).

3. Tocopherol Cyclase Reactions: Tocopherol cyclase catalyzes the formation of the 5-T from 2-methyl-6-phytylbenzoquinone and fT from 2,3-dimethyl-6-phytylbenzoquinone (67, 68) (Figure 1.7). Tocopherol cyclase from Anabaena variabilis (Cyanobacteria) blue-green algae was studied in depth by Stocker et al. (68-70). Substrate specificity is imparted through recognition of the -OH group at C-1

of the hydroquinone, the (E) configuration of the double bond on the side chain, and the length of the side chain on 2-methyl-6-phytylbenzoquinol or 2,3-dimethyl-6-phytylbenzoquinol. Substrates enter the active site of tocopherol cyclase with the recognition of the hydrophobic tail. The enzyme is equally effective in converting 2,3-dimethyl-6-geranylbenzoquinol to /-T3 and 2-methyl-6-geranylgeranyl benzoquinol to S-T3 (68). (Figure 1.8).

4. Y-Tocopherol Methyltransferase Reaction: 7-Tocopherol and S-T are methylated by a specific 7-T methyltransferase at the 5 position of the chromanol ring to yield a-and 0-T, respectively. a-And methyltransferases have not been identified in nature, and a- and f-T chromanol ring to yield a- and f-T, respectively. a- And f-are considered terminal products of the biosynthesis (67). The enzyme was purified and characterized from spinach chloroplasts and Euglena gracilis (71-73). Shintani and DellaPenna (74). showed that Y-T methyltransferase is a primary determinant of the tocopherol composition of seed oils. The Vmax values of 7-T methyltransferase from peppers were similar for 7-, S-T, and 7-, S-T3, but f-T is not a substrate (75). Overexpression of the y-T methyltransferase gene in Arabidopsis sp. increased a-T content of the oil without decreasing total tocopherol content. A seed of lines overexpressing the largest amount of Y-T methyltransferase had more than 95% of total tocopherol as a-T. To accomplish the preceding research, Arabidopsis sp. was transformed with the pDC3-A.t. y-T methyltransferase expression construct containing the Arabidopsis sp. y-T methyltransferase complementary deoxyribonucleic acid (cDNA) driven by the carrot DC3 promoter (71). Understanding of the role and activity of the y-T methyltransferase explains why many seed oils contain low a-T levels. Biosynthesis of Tocotrienols. Condensation of homogentisic acid with geranylgeranyl diphosphate, catalyzed by geranylgeranyltransferase, yields 2-methyl-6-geranylgeranyl benzoquinol, providing the substrate for formation of the tocotrienols (Figure 1.8). The 2-methyl-6-geranylgeranyl benzoquinol intermediate is converted to the respective tocotrienols by action of 2-methyl-6-phytylbenzoquinol methyltransferase. In 2002, investigation of the Y-T methyltransferase from pepper fruits indicated that methylation capacity of S- and Y-T3 is almost equivalent to the capacity to methylate the corresponding tocopherols (75). It has been postulated that enzymes participating in tocopherol and tocotrienol biosynthesis after the phytyltransferase and geranylgeranyltransferase reactions utilize both the phytylated and geranylgeranylated substrates (64, 75).

FIGURE 1.8 Conversion of homogentisic acid to tocotrienols by the action of geranylgeranyl-transferase, methyltransferase, and tocopherol cyclase. y-T, y-tocopherol; y-T3, y-tocotrienol; a-T3, a-Tocotrienol. (Modified from Refs. 67, 68.) Increasing «-Tocopherol Levels in Plant Foods. The more complete understanding of the biosynthetic steps leading to the synthesis of a-T levels in plant foods and the availability of cloned genes of the responsible enzymes have set the stage to increase a-T levels in plant foods. Thus, the nutritional impact of such foods as sources of vitamin E for the human can be increased. Approaches to engineering plants to increase concentration of a-T have been reviewed by Hess (56), Grusak and DellaPenna (76), Hirschberg (77), and DellaPenna (78). Tocopherol biosynthetic enzymes were classified into two groups by Grusak and DellaPenna:

1. Enzymes that predominantly affect quantitative aspects of the pathway (formation and phytylation of homogentisic acid)

2. Enzymes that predominantly affect qualitative aspects of the biosynthesis (cyclization and methylation enzymes)

DellaPenna (78) emphasized that research to improve the nutritional quality of plants is limited by a lack of knowledge of plant metabolism. Because of the breadth of the area, meaningful research requires an interdisciplinary effort involving nutritional biochemistry, food science, plant science, and genetics with expertise in human, animal, and plant molecular biological characteristics. Classical biochemical and genetic approaches to plant improvement are being combined with genomic approaches and rapidly developing molecular biology techniques to help identify genes of plant secondary metabolism pathways significant to improvement of nutritional quality (78). DellaPenna (78) has defined nutritional genomics as the interface between plant biochemistry, genomics, and human nutrition.

y-Tocopherol methyltransferase that converts y-T to a-T was considered to be a good molecular target to have a positive impact on a-T levels in plants. Successful manipulation of the y-T methyltransferase was achieved in Arabidopsis sp (71). Overexpression of the y-T methyltransferase gene shifted oil composition strongly toward a-T. Seeds of the lines overexpressing the gene contained as much as 80 times greater a-T concentrations when compared to normal seeds. Up-regulation of the y-T methyltransferase was, therefore, proved to be a viable approach to increasing a-T levels in plant foods. The work should be transferable to other oilseed crops that have f T as the primary tocopherol. Likewise, success will most likely be forthcoming on engineering plants to produce higher amounts of total tocopherol levels at the quantitative stages of the biosynthesis.

1.3.2. Biological Role of Vitamin E Vitamin E and Oxidative Stress. Vitamin E functions with other lipid- and water-soluble antioxidants to provide living systems an efficient defense against free radicals and the damage that they impart at the cellular level. Free radicals are defined as chemical species capable of independent existence that contain one or more unpaired electrons. Free radical generation occurs when organic molecules undergo homolytic cleavage of covalent bonds and each fragment retains one electron of the original bonding electron pair (79). This process produces two free radicals from the parent molecule with net negative charges with the ability to react with an electron of opposite spin from another molecule. Free radical generation also occurs when a nonradical molecule captures an electron from an electron donating molecule. During normal metabolism, a wide array of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced (80). The ROS and RNS include both radicals and oxidants capable of generation of free radicals (81, 82) (Table 1.4). Oxidants and oxygen radicals formed from triplet oxygen by reaction with other radicals or by photoexcitation, metabolic reactions, irradiation, metal catalysis, or heat are the primary prooxidants that induce oxidative stress in living systems or initiate autoxidative events in raw and processed foods. Reactive nitrogen species, particularly nitric oxide (NO), can contribute to oxidative stress along with ROS. Nitric oxide acts as a biological messenger with regulatory functions in the central nervous, cardiovascular, and immune systems (83). Nitric oxide is synthesized by the oxidation of arginine to NO' by nitric oxide synthetase (NOS; EC The enzyme is highly active in macrophages and neutrophils, in which NO.

TABLE 1.4 Reactive Oxygen and Nitrogen Species

Reactive oxgyen species

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