Clark, Shawn M.;
Di Leo, Rosa;
Dhanoa, Preetinder K.;
Van Cauwenberghe, Owen R.;
Mullen, Robert T.;
Shelp, Barry J.
Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis γ-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate
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Media type:
E-Article
Title:
Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis γ-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate
Contributor:
Clark, Shawn M.;
Di Leo, Rosa;
Dhanoa, Preetinder K.;
Van Cauwenberghe, Owen R.;
Mullen, Robert T.;
Shelp, Barry J.
Description:
<p>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.</p>