Abstract
Gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the gamma-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae gamma-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-gamma-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-gamma-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as gamma-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of gamma-GCS-GS (total M(r) 85,000) show 32% identity and 43% similarity with E. coli gamma-GCS (M(r) 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of gamma-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.
Highlights
Glutathione (L-␥-glutamyl-L-cysteinylglycine, GSH) is the main low molecular weight thiol in many Gram-negative bacteria and in virtually all eukaryotes except those few that lack mitochondria (e.g. Entamoeba histolytica and Giardia duodenalis) [1,2,3]
In all GSH-containing species examined to date, the tripeptide is synthesized by the sequential action of ␥-glutamylcysteine synthetase
␥-Glutamylcysteine synthetase activity was determined in crude homogenates of S. agalactiae and E. coli using a dipeptide formation assay in which L-glutamate and L-␣-[14C]Aba are converted to radiolabeled L-␥-glutamyl-L-␣-Aba (L-␣-Aba is stable and frequently used as a surrogate for L-cysteine in established E. coli [8] and mammalian [9, 10] ␥-GCS assays)
Summary
Glutathione (L-␥-glutamyl-L-cysteinylglycine, GSH) is the main low molecular weight thiol in many Gram-negative bacteria and in virtually all eukaryotes except those few that lack mitochondria (e.g. Entamoeba histolytica and Giardia duodenalis) [1,2,3]. Efforts to purify ␥-GCS from S. agalactiae were partially successful but were limited by low enzyme abundance and the instability of the isolated protein. We searched for genomic sequences in S. agalactiae that were consistent with amino acid sequences in the isolated enzyme or that had homology to previously reported prokaryotic or eukaryotic ␥-GCS or GS genes. The putative S. agalactiae ␥-GCS gene was cloned into a Qiagen pQE30 His tag expression vector, and the protein was expressed in E. coli and purified to near homogeneity (ϳ98% pure). The isolated protein is the first enzyme of GSH synthesis to be identified in a Grampositive organism and is the first bifunctional ␥-glutamylcysteine synthetase-GSH synthetase (␥-GCS-GS) to be reported in any species. A similar gene occurs in several other, mostly Gram-positive bacteria including Listeria, Clostridium, Enterococcus and Pasteurella species
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