title: Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs.
abstract: A mutant of Escherichia coli, JTG10, deficient in gamma-glutamylcysteine synthetase (gamma-ECS; EC 6.3.2.2) is unable to synthesize glutathione (GSH) and is sensitive to 8-hydroxyquinoline. This phenotype was exploited for the isolation of Arabidopsis thaliana gamma-ECS cDNAs by expression cloning, and clones were selected through functional complementation by growth on 8-hydroxyquinoline. High levels of gamma-ECS activity were detectable in extracts derived from cultures of JTG10 expressing the Arabidopsis gamma-ECS open reading frame, although these complemented mutants accumulated GSH to only 10% of the wild-type level. The derived amino acid sequence constitutes a polypeptide of 59.9 kDa and shows only 44-48% similarity with previously published sequences of rat kidney, human liver, yeast, and E. coli gamma-ECS. When the gamma-ECS cDNA was used as a probe, Southern blot analysis of Arabidopsis genomic DNA revealed that it is present as a low copy number gene. Furthermore, the Arabidopsis gamma-ECS cDNA probe failed to hybridize to maize and tobacco genomic DNA at low stringency, suggesting that heterogeneity in gamma-ECS structure exists between plant species. The activity of recombinant Arabidopsis gamma-ECS was inhibited by buthionine sulfoximine and GSH, indicating that, while differences in the primary and secondary structure of gamma-ECS from different sources exist, the enzymes may have similar active site structures.
authors.affiliation: Department of Plant Sciences, University of Oxford, United Kingdom.
authors.forename: M J
authors.initials: MJ
authors.lastname: May
authors.forename: C J
authors.initials: CJ
authors.lastname: Leaver
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008322:Mammals / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D005721:Glutamate-Cysteine Ligase