BIOCHEMICAL AND MOLECULAR ANALYSIS OF SUGARCANE GLUTAMATE DECARBOXYLASE

Abstract

Glutamate decarboxylase [GAD (EC 4.1.1.15)], a g-aminobutyric acid (GABA) metabolizing enzyme is characterized in sugarcane ( Saccharum officinarum L.) Var. Co 86032. The sugarcane GAD was purified by 7.8 folds and SDS-PAGE analysis revealed presence of apparent 65 and 52 kD GAD isomers. Biochemical characterization of Sugarcane GAD revealed the Km values of 1.6 mM for L-glutamate, 2 µM for PLP, 3.5 µM for Ca +2 and 6.3 nM for CaM at a sharp optimum pH of 6.0. Ca +2 /CaM induced sugarcane GAD by 360%, however Ca +2 alone was ineffective. In absence of Ca +2 , CaM induced the activity by 150% at pH 6.0, but no such induction was found at neutral pH. Metal ion and inhibitor studies revealed that the sugarcane GAD gets induced by Co +2 , 1-10 phenanthroline and requires -SH groups. Isolation of GAD gene through cDNA yielded 1481 bp stretch of sequence occupying distant position in the phylogram of plant GADs. Further analysis confirmed the presence of a plant specific C-terminal extension of 30-amino acid lacking authentic CaM binding domain. The results indicate the presence of at least two forms of GAD in sugarcane.