Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

Abstract

Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involves condensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyltransferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarity to chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisic acid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyl diphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9 synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols without affecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernible effect on cell growth and photosynthesis.Tocopherols are a group of amphipathic compounds synthesized only by photosynthetic organisms. The best characterized and probably most important function of tocopherols is to act as recyclable chain reaction terminators of polyunsaturated fatty acid free radicals generated by lipid oxidation. Tocopherols have a well-documented role in mammals both as an essential nutrient (vitamin E) and general antioxidant (Fryer, 1993; Liebler, 1998; BrigeliusFlohe and Traber, 1999). A similar though less welldocumented antioxidant role is also proposed for tocopherols in photosynthetic organisms (Fryer, 1992; Niyogi, 1999).From a biosynthetic perspective, tocopherols are members of a large, multifunctional family of lipidsoluble compounds called prenylquinones that also include tocotrienols, plastoquinones, and phylloquinones (vitamin K 1 ). Structural features shared by all prenylquinones include hydrophobic prenyl tails of various lengths attached to aromatic head groups that can reversibly change their redox states. Tocopherols contain a chromanol head-group and lipophillic tail derived from the 20-carbon alcohol phytol, whereas plastoquinones contain a quinone head group and isoprenoid tails of 40, 45, or 50 carbons. Such structural features are essential for the diverse biochemical and physiological roles fulfilled by various prenylquinones.The committed step in the synthesis of all prenylquinones is the condensation of various aromatic precursors and prenyl-diphosphate (DP) substrates in reactions catalyzed by a small family of related polyprenyltransferases (Lopez et al., 1996). Most aromatic and prenyl-DP substrates are utilized by more than one polyprenyltransferase (Fig. 1). For example, the aromatic compound homogentisic acid (HGA) is used for condensation with phytyl DP (PDP), geranylgeranyl DP (G...

Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana

“…Transgenic plants and mutants with altered expression of different enzymes of tocopherol biosynthesis have been created and tested under stress conditions, and represent new tools with which to investigate regulation of and by tocopherols (e.g. Shinatani and DellaPenna 1998;Tanaka et al 1999;Collakova and DellaPenna 2001;Graßes et al 2001). Transgenic plants with altered amounts of TAT are underway and are currently being tested in order to elucidate the role of TAT in tocopherol biosynthesis and the role of tocopherols under stress conditions.…”

AuaA, a Membrane‐Bound Farnesyltransferase from Stigmatella aurantiaca, Catalyzes the Prenylation of 2‐Methyl‐4‐hydroxyquinoline in the Biosynthesis of Aurachins

“…For example, at the amino acid level AuaA shares sequence similarities of 34.5 % with MenA (biosynthesis of menaquinone), [11] and 25.3 % with UbiA (biosynthesis of ubiquinone), [10] both from E. coli. AuaA also shows sequence similarities to membrane-bound prenyltransferases from plants, for example, 27.1 % to HPT from Arabidopsis thaliana [28] and 26.7 % to LePGT1 from Lithospermum erythrorhizon. [29] For expression in E. coli, auaA was amplified from genomic DNA and cloned in pQE60 to result in the expression construct pES19.…”

New insights into the function of tocopherols in plants

“…However, Synechocystis mutants with impaired tocopherol biosynthesis survive under both optimal and high light conditions in the absence of tocopherols (Collakova and DellaPenna 2001;Da¨hnhardt et al 2002). In another study, an A. thaliana mutant totally devoid of tocopherols, vte1, showed a growth reduction of only 10-15% and small reductions in chlorophyll content and photosynthetic quantum yield when exposed to a light intensity of 850 lmol photons m )2 s )1 (Porfirova et al 2002).…”

“…However, only tocopherol biosynthesis is impaired by a defect in the hpd gene in Synechocystis, thus suggesting an alternative plastoquinone biosynthetic pathway in this cyanobacterium . Moreover, it has been shown that different polyprenyltransferases are required for the synthesis of tocopherols and plastoquinones both in cyanobacteria and higher plants (Collakova and DellaPenna 2001;Schledz et al 2001;Savidge et al 2002). After the prenylation of homogentisic acid, three further enzymatic modifications occur, including two methylation reactions and a cyclization reaction forming the chromanol head group (Porfirova et al 2002).…”