Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-10, Vol.91 (21), p.10059-10063
• Main Authors: May, M.J. (Universiteit Gent, Gent, Belgium), Leaver, C.J
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN; Amino Acid Sequence; Amino acids; Animals; Arabidopsis - enzymology; ARABIDOPSIS THALIANA; Base Sequence; Blotting, Southern; Cloning, Molecular; Complementary DNA; Consensus Sequence; Corn; DNA; DNA probes; Enzymes; ESCHERICHIA COLI; Escherichia coli - enzymology; GENERO HUMANO; Genomics; GENRE HUMAIN; Glutamate-Cysteine Ligase - chemistry; Glutamate-Cysteine Ligase - genetics; Glutamate-Cysteine Ligase - metabolism; Humans; Kidney - enzymology; Kinetics; LIGASAS; LIGASE; Liver - enzymology; Mammals - metabolism; Molecular Sequence Data; Plants; Plasmids; RAT; RATA; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; SECUENCIA NUCLEICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEIQUE; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.21.10059

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