Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase

Chien Hua Pai, Bing Yu Chiang, Tzu Ping Ko, Chia Cheng Chou, Cheong Meng Chong, Fang Jiun Yen, Shoujun Chen, James K. Coward, Andrew H.J. Wang, Chun Hung Lin

Research output: Contribution to journalArticlepeer-review

51 Citations (Scopus)


Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis - amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

Original languageEnglish
Pages (from-to)5970-5982
Number of pages13
JournalEMBO Journal
Issue number24
Publication statusPublished - Dec 13 2006
Externally publishedYes


  • Binding site
  • Glutathionylspermidine
  • Mechanism
  • Structure
  • Trypanothione

ASJC Scopus subject areas

  • General Neuroscience
  • Molecular Biology
  • General Biochemistry,Genetics and Molecular Biology
  • General Immunology and Microbiology


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