title: Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis gamma-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate.
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.
authors.affiliation: Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Clark
authors.forename: Rosa
authors.initials: R
authors.lastname: Di Leo
authors.forename: Preetinder K
authors.initials: PK
authors.lastname: Dhanoa
authors.forename: Owen R
authors.initials: OR
authors.lastname: Van Cauwenberghe
authors.forename: Robert T
authors.initials: RT
authors.lastname: Mullen
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Shelp
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006038:Glyoxylates / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D021381:Protein Transport; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D006038:Glyoxylates; D011994:Recombinant Proteins; D019289:Pyruvic Acid; D000637:Transaminases; C489779:POP2 protein, Arabidopsis