Biochemical and expression analysis of gamma-aminobutyrate transaminases from Arabidopsis and tomato.

摘要

gamma-Aminobutyric acid transaminase (GABA-T) catalyzes the breakdown of GABA to succinic semialdehyde. In this thesis, the previously identified Arabidopsis thaliana (L.) Heynh GABA-T ( AtGABA-T) was characterized and three Solanum lycopersicum L GABA-Ts (Sl/GABA-T1, 2 and 3) were identified and characterized. Removal of the N-terminal targeting domains from AtGABA-T (mitochondrial), SlGABA-T1 (mitochondrial) and SlGABA-T3 (plastidial) and co-expression of all four genes with the GroES/EL molecular chaperone complex in Escherichia coli enabled recovery of soluble recombinant proteins. Activity assays indicated that the recombinant GABA-Ts have both pyruvate- and glyoxylate-dependent activities. Kinetic parameters for glyoxylate- and pyruvate-dependent AtGABA-T activities were similar, with physiologically-relevant affinities, and Lys 327 was identified as the catalytic lysine residue using site-directed mutagenesis. Assays of cell-free leaf extracts from wild-type Arabidopsis and two loss-of-function mutants in different genetic backgrounds confirmed that the native enzyme possesses both pyruvate- and glyoxylate-dependent activities. The Arabidopsis GABA-T transcript was present throughout the plant, transcript abundance increased in leaf tissue with development, and the highest transcript levels were detected in the root tissue. The three tomato GABA-T genes were expressed throughout vegetative tissue, with expression generally increasing with development and the mitochondrial form being predominant. By contrast, expression levels were much higher in reproductive tissues and the cytosolic isoform was predominant in the carpel. The dual functions of the GABA-T enzymes identified here suggest the potential for interaction between GABA metabolism and photorespiratory glyoxylate production. Furthermore, the results suggest that the GABA pathway may differ among plant species.