Cloning, biochemical and phylogenetic characterizations of gamma-glutamylcysteine synthetase from Anabaena sp. PCC 7120.
Gamma-glutamylcysteine 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 Km 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 Ki 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.
Ashida
Hiroyuki
H
Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue, Shimane, 690-8504 Japan.
Sawa
Yoshihiro
Y
Shibata
Hitoshi
H
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D014644:Genetic Variation; D005721:Glutamate-Cysteine Ligase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D005721:Glutamate-Cysteine Ligase