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Plant and Cell Physiology Advance Access originally published online on February 2, 2005
Plant and Cell Physiology 2005 46(4):557-562; doi:10.1093/pcp/pci056
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JSPP © 2005

Cloning, Biochemical and Phylogenetic Characterizations of {gamma}-Glutamylcysteine Synthetase from Anabaena sp. PCC 7120

Hiroyuki Ashida1, Yoshihiro Sawa2 and Hitoshi Shibata2,3

1 Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue, Shimane, 690-8504 Japan
2 Department of Life Science and Biotechnology, Faculty of Life and Environmental Sciences, Shimane University, Matsue, Shimane, 690-8504 Japan

3 Corresponding author: E-mail, shibata{at}life.shimane-u.ac.jp; Fax, +81-852-32-6499.

{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 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.

Received May 28, 2004; Accepted January 12, 2005
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