Plant pyruvate-dependent gamma-aminobutyrate transaminase: identification of anArabidopsiscDNA and its expression inEscherichia coli

Both pyruvate- and 2-oxoglutarate-dependent gamma-aminobutyrate transaminase (GABA-T) activities are present in crude tobacco (Nicotiana tabacum L.) leaf extracts. In this study, GABA:pyruvate-T activity was partially purified using mitochondrial isolation and protein solubilization in 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and a combination of chromatographic and electrophoretic procedures. A partial amino acid sequence of the putative 55-kDa GABA-T subunit enabled identification of a predicted Arabidopsis thaliana (L.) Heynh. GABA:pyruvate-T expressed sequence tag and subsequent amplification of a 1515 bp open reading frame encoding a 504-amino acid polypeptide. Computer analysis using web-based tools revealed the presence of a putative mitochondrial signal sequence and a pyridoxal-5-phosphate binding domain in the polypeptide. Functional expression of the GABA-T cDNA in Escherichia coli revealed that the recombinant protein uses pyruvate but not 2-oxoglutarate. The Arabidopsis GABA:pyruvate-T cDNA could form the basis for identification of multiple GABA-T isoforms and generation of GABA-T mutants for determining the fate of GABA nitrogen and elucidating the physiological function of GABA in plants.