Characterization of plant tocopherol cyclases
Tocopherols, collectively known as vitamin E, are amphiphatic molecules consisting of a polar chromanol head ring and a lipophilic isoprenoid tail. Because of the diverse functions, dietary supplements of tocopherols are thought to play an important role in improving immune function and in limiting the incidence and progression of several degenerative human diseases. Tocopherols are synthesized only in photosynthetic organisms. In plants, tocopherol biosynthesis proceeds at the inner envelope membrane of plastids. In the tocopherol biosynthetic pathway, tocopherol cyclase (TC) catalyses the key step in the biosynthesis of the chromanol substructure of the vitamin E family. To investigate the role of TC in the tocopherol biosynthesis, the present study was aimed at cloning and characterization of the TC genes from Arabidopsis and maize. The TC genes from Arabidopsis and maize were engineered for functional expression studies in E. coli. Firstly, the expression conditions were optimized to achieve the accumulation of high levels of recombinant TC proteins in the bacterial host cells. Subsequently the recombinant TC proteins, which behaved like soluble proteins and were catalytically active in their monomeric forms, were purified by affinity chromatography and used for the analysis of their enzymatic properties. These experiments revealed that the functional orthologs from the two plant species possess remarkable differences in their enzymatic properties with respect to pH and temperature optima and their kinetic constant. To gain new insights into the mechanisms regulating the tocopherol biosynthesis, chimeric TC gene constructs were overexpressed in developing seeds of transgenic rapeseed plants. This TC overexpression resulted in a significant increase in total tocopherol content, suggesting that TC activity is the limiting factor of tocopherol biosynthesis. Furthermore, overexpression of the recombinant TCs in developing seeds of Brassica napus enhanced the plastochromanol-8 content several folds. These findings show that TCs from Arabidopsis and maize possess a broad substrate specificity and can cyclize plastoquinone-9 to plastochromanol-8. In addition, they provided evidence for a regulatory function of the TC in prenyllipid metabolism.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, tocopherols, tocopherol cyclase, vitamine E
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