Characterization of glutamate decarboxylase from Synechocystis sp. PCC6803 and its role in nitrogen metabolism.

Glutamate decarboxylase (GAD) (EC 4.1.1.15), an enzyme responsible for the synthesis of γ-aminobutyric acid (GABA), from Synechocystis sp. PCC6803 was cloned and overexpressed in Escherichia coli BL21(DE3). The purified enzyme was expressed as a monomeric protein with a molecular mass of 53 and 55 kDa as determined by SDS-PAGE and gel filtration chromatography, respectively. The enzyme activity was pyridoxal-5'-phosphate dependent with an optimal activity at pH 6.0 and 30 °C. The catalytic properties of this enzyme were, Km = 19.6 mM; kcat = 100.7 s(-1); and kcat/Km = 5.1 mM(-1) s(-1). The transcription levels of genes involved in nitrogen metabolism were up-regulated in the Δgad strain. The mutant showed approximately 4- and 8-fold increases in the transcript levels of kgd and gabdh encoding a novel α-ketoglutarate decarboxylase and γ-aminobutanal dehydrogenase, respectively. Overall results suggested that in Synechocystis lacking a functional GAD, the γ-aminobutanal dehydrogenase might serve as an alternative catalytic pathway for GABA synthesis.