title: Characterization of a cold-adapted glutathione synthetase from the psychrophile Pseudoalteromonas haloplanktis.
abstract: Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-(32)P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4-8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (K(m) 0.26 mM) and γ-glutamylcysteine (K(m) 0.25 mM); a lower affinity was measured for glycine (K(m) 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (K(i) 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol(-1)) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol(-1). To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.
authors: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
authors: Antonella
authors: A
authors: Albino
authors: Salvatore
authors: S
authors: Marco
authors: Antimo
authors: A
authors: Di Maro
authors: Angela
authors: A
authors: Chambery
authors: Mariorosario
authors: M
authors: Masullo
authors: Emmanuele
authors: E
authors: De Vendittis
mesh_terms: D000222:Adaptation, Physiological; D001426:Bacterial Proteins / Q000378:metabolism*; D003080:Cold Temperature; D005978:Glutathione / Q000378:metabolism; D005981:Glutathione Synthase / Q000378:metabolism*; D031161:Pseudoalteromonas / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D005981:Glutathione Synthase; D005978:Glutathione