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Plant and Cell Physiology Advance Access first published online on February 2, 2005
This version published online on February 3, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci056
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received May 28, 2004
Revised January 11, 2005
Accepted January 17, 2005

Regular Paper

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

Hiroyuki Ashida 1, Yoshihiro Sawa 2, and Hitoshi Shibata 2*

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 & Environmental Sciences, Shimane University, Matsue, Shimane 690-8504, Japan

* To whom correspondence should be addressed.
Hitoshi Shibata, E-mail: shibata{at}life.shimane-u.ac.jp


  Abstract

{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 E. 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 mM 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.

Keywords: Anabaena-Cyanobacteria-{gamma}-Glutamylcysteine; synthetase-Glutathione-Molecular evolution.
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