Cloning, biochemical and phylogenetic characterizations of γ-glutamylcysteine synthetase from Anabaena sp PCC 7120

gamma-Glutamyleysteine synthetase (EC 6.3.2.2, gamma-GCS) catalyzes the first step of glutathione synthesis: L-Glu + L-CyS + ATP = gamma-L-glutamyl-L-cysteine (gamma-GC) + ADP + Pi. We have cloned the gene alr3351 of Anabaena sp. PCC 7120, expressed the recombinant enzyme in Escherichia coli, and characterized its product as gamma-GCS by analyzing gamma-GC production, ADP formation and Pi release. Apparent K-m values for L-Glu, ATP and L-CyS were estimated to be 0.82, 0.23 and 0.14 mM, respectively. Glutathione and L-buthionine sulfoximine were inhibitors with K-i values of 6.5 and 29.3 mM, respectively. The molecular mass of Anabaena gamma-GCS was estimated to be 43.4 kDa by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The important sequence for the activity of plant gamma-GCS was found in alpha-proteobacterial gamma-GCSs but not in cyanobacterial enzymes, suggesting that the cyanobacterial gamma-GCS gene is not the primary progenitor for the plant genes.