Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis γ-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate

Abstract

γ-Aminobutyrate transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, the previously identified Arabidopsis thaliana (L.) Heyhn GABA-T (AtGABA-T) was characterized in more detail. Full-length AtGABA-T contains an N-terminal 36 amino acid long targeting pre-sequence (36 amino acids) that is both sufficient and necessary for targeting the enzyme to mitochondria. Removal of the pre-sequence encoding this N-terminal targeting domain and co-expression of the resulting truncated AtGABA-T cDNA with the GroES/EL molecular chaperone complex in Escherichia coli yielded good recovery of the soluble recombinant proteins. Activity assays indicated that purified recombinant GABA-T has both pyruvate- and glyoxylate-dependent activities, but cannot utilize 2-oxoglutarate as amino acceptor. Kinetic parameters for glyoxylate- and pyruvate-dependent GABA-T activities were similar, with physiologically relevant affinities. Assays of GABA-T activity in cell-free leaf extracts from wild-type Arabidopsis and two knockout mutants in different genetic backgrounds confirmed that the native enzyme possesses both pyruvate- and glyoxylate-dependent activities. The GABA-T transcript was present throughout the plant, but its expression was highest in roots and increased as a function of leaf development. A GABA-T with dual functions suggests the potential for interaction between GABA metabolism and photorespiratory glyoxylate production.

Targeted Enhancement of Glutamate-to-γ-Aminobutyrate Conversion in Arabidopsis Seeds Affects Carbon-Nitrogen Balance and Storage Reserves in a Development-Dependent Manner

“…The thickness of the TCA cycle circular scheme refers to the reduced TCA cycle activity during seed desiccation. Clark et al (2009) suggest that 2-OG-dependent GABA-T activity is not present in plants. In italics are given the genes associated with metabolic processes whose expression was significantly changed in the transgenics (Supplemental Tables S3 and S4).…”

“…It is possible that GABA-induced amino acid biosynthesis occurs via the TCA cycle, as suggested by our labeling experiments. Alternatively, accumulating GABA could be transaminated by GABA-T to produce Ala from pyruvate, or Gly from glyoxylate to Gly (Clark et al, 2009), thus contributing to amino group incorporation into the C skeleton and partly explaining the depletion of intermediates of C metabolism and fatty acid biosynthesis. This interpretation receives support from the up-regulation of Ala-glyoxylate transaminase.…”

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase

“…Full-length or N-terminally truncated At5g32470 cDNA, or the predicted mitochondrial targeting sequence, were cloned into the NcoI site of pUC18/ NcoI-mGFP, immediately 59 of the open reading frame for monomeric GFP (Clark et al, 2009) to create peptide-mGFP fusions. The targeting regions that were removed, or fused directly to GFP, are shown in Figure 7.…”

Structure and Regulation of thegabGene Cluster, Involved in the γ-Aminobutyric Acid Shunt, Are Controlled by a σ54Factor inBacillus thuringiensis

“…Recent findings suggest that GABA has a role as a signal molecule in plant development as well (9,13,41). The study of the GABA shunt in animals and plants is still very active (2,14,20,36), but the biological functions of the GABA shunt and its metabolites in bacteria, particularly Bacillus sp., are less clear. GABA was found to act as a germinant for Bacillus megaterium spores (21).…”