Gaba: Pyruvate-dependent transaminase dominates gaba: 2-oxoglutarate dependent transaminase in sugarcane and their molecular characterization

GABA occupies a significant component of cytosolic free amino acid pool.  GABA-Transaminase (GABA-T, EC 2.6.1.19), the second enzyme of GABA shunt, converts GABA to Succinic semialdehyde (SSA). GABA pyruvate- (GPT) and 2-oxoglutarate- (GOT) dependent gamma-aminobutyrate transaminase (GABA-T) activities were noted in crude leaf extract of sugarcane var. Co 86032.  The enzyme was partially purified by DEAE-Cellulose and Sephadex G-100 columns. Non-denaturing PAGE analysis of ~90 folds purified GABA-T showed 65, 60 and 55 kD proteins, which were further confirmed by activity staining. GPT and GOT forms were active at pH 6.2 and 8.2 respectively. The Km values of both forms were determined.  Sugarcane GPT was found to prefer beta-alanine and ornithine to GABA but GOT was specific to 2-oxoglutarate.  GOT was induced in presence of Cu+2 and GPT in presence of Fe+3 while proline and succinic semialdehyde inhibited both forms. Sugarcane GABA-T is a pyridoxal 5’-phosphate (PLP) dependent enzyme. A 1458 bp GABA-T gene was isolated and characterized by comparing with the gene sequences from other sources.  A 463 amino acid long deduced protein possessed mitochondrial sorting signal but PLP binding site was missing.