Isolation and Identification of Genes in the Vitamin E Biosynthetic Pathway

The biosynthetic pathway of vitamin E was elucidated several years ago, but the genes encoding the enzymes of the pathway have been identified only very recently (Table 18.2). Vitamin E biosynthetic enzymes are found in chloroplasts [40], [50-53] and chromoplasts [54].

The first potential enzymes involved in tocopherol synthesis are the hydrox-yphenyl pyruvate dioxygenase (HPPD) and geranylgeranyl diphosphate reductase (GGDPR). A single copy of the gene encoding the HPPD was identified in Dau-cus carota, Arabidopsis thaliana, barley (Hordeum vulgare), and Synechocystis sp. PCC 6803 and is essential for both tocopherol and plastoquinone biosynthesis [26, 28, 45, 55-57]. HPPD is a cytosolic enzyme [64]. HPPD and GGDPR were selected as candidate regulatory enzymes based on in vivo observations showing that HGA and PDP might be limiting factors in tocopherol biosynthesis. Tissue-culture studies have shown that exogenously supplied HGA and PDP both caused significant increases in tocopherol biosynthesis [58]. The expression of gene-encoding HPPD, which is overexpressed in the leaves and seeds of both Arabidopsis and tobacco, proves that HGA levels limit tocopherol biosynthesis [59, 60].

In a recent patent, Grimm and Tanaka [62] identified a geranylgeranyl reductase cDNA (CHL P) from a Lambda ZAP II cDNA library of tobacco. The enzyme of geranylgeranyl reductase is involved in isoprenoid metabolism and functions in two metabolic pathways: tocopherol biosynthesis and chlorophyll biosynthesis.

Another important flux-regulating enzyme is homogentisate phytyltransferase (HPT), encoded by the Arabidopsis vitamin E2 (VTE2) locus. The genes encoding HPT have been independently identified using bioinformatics in three different groups in Synechocystis sp. PCC 6803 and Arabidopsis based on the similarity of their sequence to chlorophyll synthases [32, 63, 64].

Table 18.2 Enzymes and genes involved in vitamin E biosynthesis from different species

Enzyme activity

Species

Gene

Locus

Substrate

Reference

Homogentisate

Synechocystis

Slr1736

HGA, PDP,

[32,63,64]

phytytransferase (HPT)

Arabidopsis

At2g18950

VTE2, HPT1

GGDP

[32, 64]

Homogentisate

Arabidopsis

HGA, PDP,

[36]

geranylgeranyl

transferase (HGGT)

Barley, rice, wheat

GGDP

[47]

Homogentisate

Arabiopsis

At3g11950

VTE2-paralog

[84, 85]

prenyldiphosphate

transferase

2-Methyl-6-

Synechocystis

SH0418

MPBQ,

[73]

prenylbenzoquinol

methyltransferase

Arabidopsis

At3g63410

VTE3

MGGBQ

[34, 74]

(PrBQMT)

Tocopherol/tocotrienol

Sunflower

(D)MPBQ,

[86]

cyclase

(D)MGGBQ

(TC)

Synechocystis

Slr1737

[77]

Arabidopsis

At4g32770

VTE1

[33,77]

Tocopherol/tocotrienol

Maize

SXD1

§-,y-

[78]

methyltransferase (TMT)

Potato

StSXD1

Tocopherol, [51]

Synrchocystis

Slr0089

§-,y-

[29]

Arabidopsis

At1g64970

VTE4

Tocotrienol

[29, 87]

Phytol kinase (PK)

Perilla

Slr1652

Phytol,

[88]

Sunflower

At5g04490

VTE5

PMP

[89]

Synechocystis

[90]

Arabidopsis

[90]

P-Hydroxypherylpyruvic

Synechocystis

Slr0090

HPP

[28, 45, 56]

acid

dioxygenase (HPPD)

Arabidopsis

At1g06590

PDS1

Homogentisic acid geranylgeranyl transferase (HGGT) is a functionally divergent form of HPT that displays substrate specificity for GGDP in preference to PDP. cDNAs encoding HGGT, with 40 to 50% identity to Arabidopsis HPT, were isolated from seeds of monocot species, such as barley, wheat (Triticum aestivum), and rice (Oryza sativa) [47]. Tocotrienols are not always synthesized by dicot species, as observed by Cahoon et al. [47]. When the barley HGGT gene was overexpressed in Arabidopsis leaves, tocotrienols accumulated in very high levels, whereas to-copherol levels were not affected. This indicates that HGGT and HPT are highly specific for their prenyl substrates, GGDP and PDP respectively, and must compete with one another for HGA. Although overproduction of tocotrienols clearly shows that GGDP and HGA are abundantly available, the fact that HGGT overexpression has no effect on tocopherol production indicates that PDP must be limiting. Therefore, overexpression of GGDP reductase could increase PDP availability significantly and, thus, increase pathway flux.

The role of GGH in catalyzing the conversion of GGDP to PDP in tocopherol and chlorophyll biosynthesis has been suggested [10-13]. Studies on Synechocystis GGH deletion mutants and antisense expression of GGH in tobacco plants clearly demonstrate that GGH is essential for tocopherol biosynthesis in bacteria and plants [65-67]. However, transgenic expression of At-GGH alone, or co-overexpression of At-GGH with At-HPPD, Eh-TYRA, and At-VTE2 in soybean seeds, did not reduce tocotrienol content [68], suggesting that an additional independent pathway may govern the synthesis of PDP in plants. This was confirmed by early reports of the presence of strong phytol kinase activity in spinach leaf chloroplasts and a recent Arabidopsis mutant encoding a phytol kinase gene [40, 69]. The gene of phytol kinase is essential for the biosynthesis of at least 80% of the seed tocopherols in Arabidopsis [70], suggesting that 80% of PDP for tocopherol biosynthesis is catalyzed by phytol kinase and up to 20% of PDP is formed directly via reducing GGDP.

The activity of MPBQ/MSBQ MT has been detected in spinach (Spinacia oleracea) chloroplasts [41], and maize (Zea mays) and sunflower (Helianthus an-nuus) mutants have been identified with phenotypes that disrupt MPBQ/MSBQ MT activity [71, 72]. MPBQ/MSBQ MT was cloned from Synechocystis PCC6803 (SLL0418) based on the similarity of its sequence to y-TMT [34, 73]. Because the plant ortholog of SLL0418 was not functional, MPBQ/MSBQ MT was cloned from Arabidopsis (VTE3) via the map-based cloning approach using partial loss-of-function alleles from EMS mutagenized Arabidopsis plants [34, 74]. Interestingly, the two proteins of VTE3 and SLL0418 shared less than 20% amino acid identity but displayed similar activities in the tocopherol and plastoquinone pathways [34, 74], suggesting that the convergent evolution occurred in this step of the pathway in cyanobacteria and plants. VTE3 is the only enzyme of the pathway that is also involved in plastoquinone synthesis, which catalyzes a key methylation step in both tocopherol and plastoquinone (PQ) synthesis [34, 73-75]. The essential function of VTE3 in two independent metabolic pathways can increase the challenge of to-copherol pathway engineering; as altered tocopherol intermediate pools, resulting from enhanced tocopherol flux, can lead to substrate competition between MPBQ or MGGBQ and the plastoquinol precursor MSBQ.

The function of tocopherol cyclase (TC) in tocopherol biosynthesis was discovered with the help of mutational analysis in Synechocystis [76, 77]. TC, encoded by the Arabidopsis VTE1 loci, catalyzes the formation of the chromanol headgroup of the various tocopherol isoforms, DMPBQ or MPBQ. So far, TC has been purified and characterized only from the cyanobacterium Anabaena variabilis [79]. Recently, genes coding for the TC from Synechocystis sp. PCC6803 (slr1737), Arabidopsis thaliana (VTE1), and maize (SXD1) have been cloned [33, 77, 78, 80]. The mutation of genes VTE1, SXD1, or slr1737 resulted in both a tocopherol deficiency and an accumulation of 2,3-dimethyl-6-phytyl-1,4-benzoquinone (DMPBQ), which suggests that TC activity is evolutionarily conserved between plants and cyanobacteria.

The y-tocopherol methyltransferase (y-TMT), encoded by a VTE2 locus in the Arabidopsis gene, was also discovered through bioinformatics analysis and confirmed by mutational analysis in Synechocystis [29]. A genomics-based approach was used to clone y-TMT, which is the final enzyme in a-tocopherol synthesis.

The gene for y-TMT in the Synechocystis PCC6803 genomic database was identified using Arabidopsis HPPDase to initiate a search. To identify the y-TMT gene from Arabidopsis, the Synechocystis y-TMT protein sequence was used to search the Arabidopsis expressed sequence tag (EST) database, resulting in the discovery of a cDNA clone with a 66% amino-acid-sequence similarity with Synechocystis [29,81].

The isolation and identification of the enzymes in the vitamin E biosynthetic pathway has driven researchers to bioengineer plants with improved vitamin E content and alterations in its composition. Meanwhile, quantitative vitamin E loci in Arabidopsis provide insight into the regulation and/or metabolism of vitamin E in plants and has clear ramifications for improving the nutritional content of crops through marker-assisted selection and metabolic engineering [82].

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