Charakterisierung der Branched-Chain Aminotransferasen BCAT1 und BCAT6 und der Isopropylmalat Isomerase in Arabidopsis thaliana
Auch gedruckt in der BibliothekW: W-H 14.241
FakultätFakultät für Naturwissenschaften
Ressourcen- / MedientypDissertation, Text
Datum der Freischaltung2015-07-03
Arabidopsis expresses 6 branched-chain amino transferases (BCAT). The Studies showed BCAT6 to be located in cytosol. Recombinant BCAT6 shows activity with BCAAs, Met and their 2 oxoacids. GUS-assays suggested activity of the BCAT6 promoter near the vascular bundles. The quantification of amino acids and glucosinolates in bcat6 1 single, bcat4 2 x bcat6 1 double and bcat3 1 x bcat4 2 x bcat6 1 triple mutants indicated a function of BCAT6 in the deamination of Met during biosynthesis of methionine-derived glucosinolates. Further, the analysis of BCAT1 indicated this protein to be located in mitochondria. The knockout of BCAT1 in Arabidopsis leads to an enrichment of all three BCAAs. This indicates a function in the initial step during degradation of BCAAs. In addition, the loss of BCAT1 leads to decreases of carbohydrates, succinic and fumaric acid. This indicates that BCAA degradation supports the recovery of energy via the TCA-cycle. Further results indicated that the degradation of BCAAs contributes to recycling of nitrogen by recovery of Gly and Gln. A further goal of this thesis was to investigate the specific features of the 3 small subunits (SSU) of the Arabidopsis isopropylmalate isomerase (IPMI) which contribute to the functional specification of the enzyme. The 3 subunits were each fused to different fluorescent proteins and were expressed under control of their native promoters. Microscopic analysis revealed SSU1 to be localized in plastids in the cortex of roots and in the epidermis of leaves. SSU2 and -3 were co-localized in cells close to xylem or phloem. To test whether the amino acid sequences in the SSUs can contribute to functional specification of IPMI, the substrate recognition site (SRR) of SSU1 was replaced by the SRR of SSU3. This chimeric protein was expressed in the double mutant ipmi ssu2 1 x ipmi ssu3 1. The analysis of glucosinolates in the complemented mutant revealed a specialization of the SSUs caused by different amino acid structures.
Branched-chain amino acids
Freie SchlagwörterBranched-Chain Aminotransferase