title: Gamma-glutamylcysteine synthetase-glutathione synthetase: domain structure and identification of residues important in substrate and glutathione binding.
abstract: In most organisms, glutathione (GSH) is synthesized by the sequential action of distinct enzymes, gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase (GS). In Streptococcus agalactiae, GSH synthesis is catalyzed by a single enzyme, gamma-glutamylcysteine synthetase-glutathione synthetase (gamma-GCS-GS). The N-terminal sequence of gamma-GCS-GS is similar to Escherichia coli gamma-GCS, but the C-terminal sequence is an ATP-grasp domain more similar to d-Ala, d-Ala ligase than to any known GS. In the present studies, C-terminally and N-terminally truncated constructs were characterized in order to define the limits of the gamma-GCS and GS domains, respectively. Although WT gamma-GCS-GS is nearly uninhibited by GSH (K(i) approximately 140 mM), shorter gamma-GCS domain constructs were unexpectedly found to be strongly inhibited (K(i) approximately 15 mM), reproducing a physiologically important regulation seen in monofunctional gamma-GCS enzymes. Because studies with E. coli gamma-GCS implicate a flexible loop region in GSH binding, chimeras of S. agalactiae gamma-GCS-GS were made containing gamma-GCS domain flexible loop sequences from Enterococcus faecalis and Pasteurella multocida gamma-GCS-GS, isoforms that are inhibited by GSH. Inhibition remained S. agalactiae-like (i.e., very weak). C-Terminal constructs of gamma-GCS-GS have GS activity (0.01-0.04% of WT), but proper folding and significant GS activity required a covalently linked gamma-GCS domain. In addition, site-directed mutants in the middle region of the gamma-GCS-GS sequence established that GS activity depends on residues in a region that is also part of the gamma-GCS domain. Our results provide new insights into the structure of gamma-GCS-GS and suggest gamma-GCS-GS evolved from a monomeric gamma-GCS that became C-terminally fused to a multimeric ATP-grasp protein.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Blythe E
authors.initials: BE
authors.lastname: Janowiak
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hayward
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
authors.forename: Owen W
authors.initials: OW
authors.lastname: Griffith
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004151:Dipeptides; D013293:Enterococcus faecalis / Q000201:enzymology*; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry* / Q000235:genetics; D005978:Glutathione / Q000737:chemistry*; D005981:Glutathione Synthase / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D004151:Dipeptides; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione; C017341:gamma-glutamylcysteine