title: Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.
abstract: 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.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, No. 128 Academia Road Section 2, Nan-Kang, Taipei 11529, Taiwan.
authors.forename: Chien-Hua
authors.initials: CH
authors.lastname: Pai
authors.forename: Bing-Yu
authors.initials: BY
authors.lastname: Chiang
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.forename: Chia-Cheng
authors.initials: CC
authors.lastname: Chou
authors.forename: Cheong-Meng
authors.initials: CM
authors.lastname: Chong
authors.forename: Fang-Jiun
authors.initials: FJ
authors.lastname: Yen
authors.forename: Shoujun
authors.initials: S
authors.lastname: Chen
authors.forename: James K
authors.initials: JK
authors.lastname: Coward
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
authors.forename: Chun-Hung
authors.initials: CH
authors.lastname: Lin
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019732:Amide Synthases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017385:Sequence Homology; D013095:Spermidine / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010710:Phosphates; D000255:Adenosine Triphosphate; D019732:Amide Synthases; C081536:glutathionylspermidine synthetase; D005978:Glutathione; D008274:Magnesium; D013095:Spermidine