Identifizierung und Charakterisierung von Homogentisat-Prenyltransferasen aus dem Tocopherol- und dem Plastochinon-9-Biosyntheseweg

Tocopherols and plastoquinone-9 are characteristic prenyllipids of photosynthetic organisms. In plants, the first committed step in the biosynthesis of these compounds is catalyzed by homogentisate prenyltransferases located in the inner envelope membranes of chloroplasts. These enzymes use homogentisate derived from the shikimate pathway and prenyl diphosphates synthesized via the plastidial non-mevalonate isoprenoid pathway as substrates and catalyze the decarboxylation and prenylation of homogentisate. The condensation of homogentisate and phytyl diphosphate provides the precursor for the formation of tocopherols, while prenylation with solanesyl diphosphate results in plastoquinone-9 biosynthesis. Within the scope of the present thesis, four prenyltransferase sequences were cloned, one from Synechocystis sp., two cDNAs from Arabidopsis thaliana and one cDNA from Chlamydomonas reinhardtii. The sequences encode hydrophobic proteins with several putative transmembrane domains. For the eucaryotic proteins, N-terminal plastidial transit peptides were predicted. The alignment of the amino acid sequences revealed conserved prenyltransferase motifs. In addition, these analyses showed that one Arabidopsis protein possesses higher homology to the Synechocystis enzyme whereas the other shares highest similarities with the Chlamydomonas enzyme. By functional expression studies in Escherichia coli, the catalytical activities of these enzymes in bacterial membranes were demonstrated and the prenyltransferases were further characterized. All enzymes accepted homogentisate as substrate but they differed in their prenyl diphosphate specificities. The Synechocystis enzyme as well as one Arabidopsis prenyltransferase specifically utilized phytyl diphosphate as prenyl donor and thus catalyzed the first step in tocopherol biosynthesis. In contrast, the Chlamydomonas enzyme and the second Arabidopsis enzyme reached highest activities with solanesyl diphosphate and were hardly active with phytyl diphosphate indicating that these prenyltransferases are involved in plastoquinone-9 biosynthesis. The in vivo function of the Synechocystis enzyme was confirmed by the development and analysis of a Synechocystis mutant in which the homogentisate prenyltransferase gene was disrupted by the insertion of an antibiotic resistance gene leading to complete inactivation of tocopherol synthesis. In order to study the effects of eucaryotic prenyltransferases on prenyllipid biosynthesis, transgenic plants expressing the Arabidopsis cDNAs were developed. The results are consistent with the substrate specificities determined by enzymic assays. Expression of the homogentisate phytyltransferase gene in combination with genes for hydroxyphenylpyruvate dioxygenase and cyclase resulted in a 2.4 fold increase of tocopherol levels in seed oils of transgenic rapeseed plants in comparison to wild type plants. Overexpression of the second Arabidopsis gene in transgenic Arabidopsis plants led to a significant increase in plastoquinone-9 content but stimulated probably via indirect mechanisms tocopherol biosynthesis, too. Hence, these data provided further indications for a close interaction between the prenyllipid biosynthesis pathways in plants