Journal Article

Cloning, Biochemical and Phylogenetic Characterizations of γ-Glutamylcysteine Synthetase from <i>Anabaena</i> sp. PCC 7120

Hiroyuki Ashida, Yoshihiro Sawa and Hitoshi Shibata

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 4, pages 557-562
Published in print April 2005 | ISSN: 0032-0781
Published online April 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci056
Cloning, Biochemical and Phylogenetic Characterizations of γ-Glutamylcysteine Synthetase from Anabaena sp. PCC 7120

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γ-Glutamylcysteine synthetase (EC 6.3.2.2, γ-GCS) catalyzes the first step of glutathione synthesis: l-Glu + l-Cys + ATP = γ-l-glutamyl-l-cysteine (γ-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 γ-GCS by analyzing γ-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 γ-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 γ-GCS was found in α-proteobacterial γ-GCSs but not in cyanobacterial enzymes, suggesting that the cyanobacterial γ-GCS gene is not the primary progenitor for the plant genes.

Keywords: Anabaena; Cyanobacteria; γ-Glutamylcysteine synthetase; Glutathione; Molecular evolution; ABA, l-α-aminobutyric acid; BSO, l-buthionine sulfoximine; γ-GC, γ-glutamylcysteine; γ-GCS, γ-glutamylcysteine synthetase (EC 6.3.2.2); GSH-II, glutathione synthetase (EC 6.3.2.3); GSH, reduced form of glutathione; IPTG, isopropyl-β-d-thiogalactopyranoside; ORF, open reading frame; PCC, Pasteur Culture Collection

Journal Article.  4033 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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