Catabolism of GABA in apple fruit: Subcellular localization and biochemical characterization of two γ-aminobutyrate transarninases

gamma-Aminobutyrate (GABA) accumulates in apple fruit during controlled atmosphere storage. Here, we demonstrated that GABA levels declined markedly in apples (Maus x domestica Borkh. cv. Empire) within 3 h after transfer from controlled atmosphere storage (3 degrees C, 2.5 kPa O-2, 2.5 kPa CO2) to ambient conditions. Also, we identified two genes encoding apple fruit GABA transaminase (GABA-T), the enzyme responsible for the catabolism of GABA to succinic semialdehyde. The deduced amino acid sequences of the two MdGABA-T enzymes were 93% identical to each other, and 74-83% identical to known Arabidopsis and tomato GABA-Ts. Transient expression of the individual full-length proteins fused to the green fluorescent protein in tobacco suspension-cultured cells revealed that MdGABA-T1 and MdGABA-T2 were localized to mitochondria. Removal of the N-terminal targeting presequences yielded good recovery of the soluble recombinant proteins in Escherichia coli when they were co-expressed with the GroES/EL molecular chaperone complex. Continuous monitoring of recombinant GABA-T activity via a bacterial NADP(+))-dependent succinic semialdehyde dehydrogenase-linked assay established that the two GABA-Ts in apple fruit, like the mitochondrial GABA-T in Arabidopsis and the mitochondrial, plastidial and cytosolic GABA-Ts in tomato, utilized pyruvate and glyoxylate, but not 2-oxoglutarate. Thus, the substrate specificity of the two apple fruit GABA-Ts was similar to that for the GABA-Ts in Arabidopsis and tomato. However, the existence of two GABA-Ts in the mitochondria of apple fruit differed from the scenarios found in the other two species, providing yet another variation on the subcellular distribution of GABA-Ts in plant cells. (c) 2012 Elsevier B.V. All rights reserved.