CHARACTERIZATION OF GLUTAMATE DECARBOXYLASE IN Synechocystis sp. PCC 6803 AND ITS ROLES IN RESPONSE TO ABIOTIC STRESS

Abstract

Glutamate decarboxylase (GAD) is a pyridoxal-5′-phosphate dependent enzyme that catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA) through irreversible decarboxylation reaction. GAD is widely distributed in nature and found to be occurring in bacteria, fungi, higher plants and mammalian central nervous system. Effect of various abiotic factors such as light (visible and UV), temperature, pH, alternative nitrogen and carbon source, osmotic stress, glutamate and polyamine supplementation on glutamate decarboxylase (GAD) activity of Synechocystis was studied by using a one-variable-at-a time approach. Synechocystis GAD showed higher activity when late log phase cells were exposed to 24 h of 40 °C temperature, osmotic stress induced by 50 mM NaCl and 100 mM sorbitol, and 60 min of UV-B irradiation. 0.2 % w/v glucose was considered as an optimum carbon source for GAD activity. GABA accumulation inside the cells of Synechocystis was observed to be increasing in response to 0.1 % glucose as carbon source, UV-B irradiation, and NaCl and sorbitol induced osmotic stress at higher concentrations. 0.5 mM spermidine and 10 mM glutamate supplementation resulted in an increase in GAD activity by 2.2 and 3.5- fold respectively, with a concomitant increase in intracellular GABA levels. Synechocystis mutant strain deficient in GAD activity, showed very low intracellular GABA and higher glutamate levels when compared to wild type strain. However increase in GABA content was observed in mutant under polyamine supplementation and UV-B irradiation, suggesting the alternative routes of GABA production in Synechocystis. Transcript levels of gene encoding GAD (gad) were observed to be affected by alternative carbon sources and short term osmotic stress conditions. Stress induced by UV-B radiation resulted in down-regulation of gad transcripts, suggesting the involvement of post translational modification of GAD enzyme. Synechocystis gad gene (sll1641) was cloned and transformed into E. coli to obtain a recombinant GAD. Activity of recombinant GAD was optimal at pH 5.8. Optimum concentration of substrate for conversion into GABA by recombinant GAD was 30 mM glutamate. Recombinant GAD was confirmed as a monomer with apparent molecular mass of 53 kDa and found to be a pyridoxal-5′-phosphate dependent enzyme. Km and Vmax values were 19.6 mM and 21.5 nmol min-1 mg-1 respectively. Altogether the results suggested that alteration in GAD activity and GABA levels under abiotic stress conditions confer a stress relieving role to Synechocystis and GAD plays an important role in connecting the C/N metabolism in Synechocystis.