|
type |
Journal Article |
authors |
Clark SM, Di Leo R, Dhanoa PK, Van Cauwenberghe OR, Mullen RT, Shelp BJ.
|
title |
Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis gamma-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate |
journal |
J Exp Bot |
Activity |
2.6.1.96 |
Family |
2.6.1.96 |
sel |
selected |
ui |
19264755 |
year |
(2009) |
volume |
60 |
number |
6 |
pages |
1743-57 |
| |
keywords |
amino acceptor, gamma-aminobutyrate, gamma-aminobutyrate transaminase, protein purification, heterologous expression, recombinant protein. |
abstract |
Gamma-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. |
last changed |
2019/01/08 09:26 |
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