title: The cold-adapted γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis.
abstract: A recombinant γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming γ-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg(++); 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15 °C was higher than that reported for rPhGshB, thus suggesting that formation of γ-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant γ-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. haloplanktis.
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Albino
authors.affiliation: Dipartimento di Bioscienze e Territorio, Università del Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italy.
authors.forename: Amalia
authors.initials: A
authors.lastname: De Angelis
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Marco
authors.affiliation: Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, II Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Severino
authors.affiliation: Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, II Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.affiliation: Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, II Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy.
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.affiliation: Dipartimento di Bioscienze e Territorio, Università del Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italy.
authors.forename: Doriana
authors.initials: D
authors.lastname: Desiderio
authors.affiliation: Dipartimento di Bioscienze e Territorio, Università del Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italy.
authors.forename: Gennaro
authors.initials: G
authors.lastname: Raimo
authors.affiliation: Dipartimento di Scienze Motorie e del Benessere, Università di Napoli "Parthenope", Via Medina 40, 80133 Napoli, Italy.
authors.forename: Mariorosario
authors.initials: M
authors.lastname: Masullo
authors.affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy. Electronic address: devendit@unina.it.
authors.forename: Emmanuele
authors.initials: E
authors.lastname: De Vendittis
mesh_terms: D000222:Adaptation, Physiological; D003080:Cold Temperature; D003545:Cysteine; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006706:Homeostasis; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D003545:Cysteine
keywords: Cold adaptation; Glutamyl-cysteine ligase; Glutathione biosynthesis; Pseudoalteromonas haloplanktis; Psychrophile