Identification and functional analyses of homogentisate phytyltransferase; A key enzyme involved in tocopherol synthesis in photosynthetic organisms

Tocopherols are lipid soluble antioxidants collectively known as Vitamin E, an essential component of the human diet. Tocopherols consist of a polar chromanol ring and a hydrophobic prenyl chain derived from homogentisate (HGA) and phytyl diphosphate (PDP), respectively. Condensation of these two compounds represents the committed step in tocopherol biosynthesis catalyzed by homogentisate phytyltransferase (HPT). HPTs in Synechocystis sp. PCC 6803 (SynHPT) and Arabidopsis thaliana (AtHPT) were identified based on their sequence similarity to chlorophyll synthases, which also utilize PDP. Both SynHPT and AtHPT showed prenyltransferase activity in vitro with HGA and PDP as preferred substrates. Neither enzyme was active with solanesyl diphosphate, the prenyl substrate used in plastoquinone-9 biosynthesis. Plastoquinone-9 levels were unaffected in Synechocystis sp. PCC 6803 HPT disruption mutant, which showed a complete absence of tocopherols. These results suggest existence of separate polyprenyltransferase activities involved tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803.; Tocopherol levels fluctuate during plant growth, possibly due to an altered expression of genes involved in tocopherol synthesis and accumulation. Because HPT catalyzes the committed step of tocopherol biosynthesis, it is likely that HPT is a regulated enzyme, activity of which may limit tocopherol synthesis in non-stressed and stressed plants. This hypothesis was addressed by generating transgenic Arabidopsis plants constitutively overexpressing AtHPT (35S:: HPT1). Increases in HPT specific activity correlated with elevated total tocopherol levels observed in transgenic lines under normal and high-light stress conditions. Non-stressed 35S::HPT1 lines accumulated a 4.4-fold and 40% increase in total tocopherol levels in leaves and seeds, respectively, relative to wild type plants. Total tocopherol levels in 35S:: HPT1 increased 2- to 3.8-fold relative to wild type during high light stress. These results indicate that HPT activity limits tocopherol biosynthesis in non-stressed and stressed wild type Arabidopsis plants. Stress resulted in an up-regulation of HPT and several other enzymes involved in tocopherol accumulation and concomitantly in a dramatic increase in total tocopherol levels in both wild type and 35S::HPT1, suggesting that HPT is not the only enzyme involved in regulation of tocopherol biosynthesis. The presence of high levels of other tocopherols in stressed plants also suggested a possible limitation of α-tocopherol synthesis by other tocopherol-related enzymes. Identification of the steps limiting tocopherol synthesis is crucial for understanding the regulation of the pathway, which can be applied in plant metabolic engineering of the tocopherol biosynthetic pathway.