The glutathione biosynthesis in the psychrophile Pseudoalteromonas haloplanktis

• Main Author: Albino, Antonella
• Format: Doctoral or Postdoctoral Thesis
• Language: Italian; English
• Published: 2011
• Subjects: Antarctic; Antarc*
• Online Access: http://www.fedoa.unina.it/8856/; http://www.fedoa.unina.it/8856/1/Albino_Antonella_24.pdf; https://doi.org/10.6092/UNINA/FEDOA/8856

Reduced glutathione (GSH), together with its oxidized form (GSSG), is the most effective antioxidant system responsible for controlling the cellular redox state. Its biosynthesis from glutamate, cysteine and glycine, normally requires two enzymes. Indeed, γ-glutamyl-cysteine ligase (GshA) forms γ-glutamyl-cysteine, whereas glutathione synthetase (GshB) leads to the formation of GSH. In the genome of Pseudoalteromonas haloplanktis, a psychrophilic eubacterium isolated from Antarctic sea water, two genes coding for GshA (PhGshA-I and PhGshA-II ) and one gene for GshB (PhGshB) were putatively identified. The study of the biochemical properties of these enzymes was addressed with an appropriate heterologous expression system, thus leading to the production of the recombinant forms of PhGshB and PhGshA-II (rPhGshB and rPhGshA-II), purified by affinity chromatography. The first enzyme investigated was rPhGshB. Its purification was achieved either in the absence or in the presence of β-mercaptoethanol. The study of its molecular properties showed that, when purified in the presence of β-mercaptoethanol, rPhGshB underwent a covalent modification; however, this modification did not significantly affect its biochemical properties. The molecular mass of rPhGshB in denaturing conditions was 36 kDa, corresponding to the mass of the PhGshB monomer; in non denaturing conditions the mass determined by gel-filtration ranged between 74 and 136 kDa, respectively, values corresponding to a dimeric or tetrameric organization. The different behavior depended on the enzyme concentration and the data suggested that at higher concentrations the enzyme formed an unstable tetramer that at lower concentrations was converted into a dimeric and more stable form. To study the activity of rPhGshB, a new method for direct determination was developed, based on the hydrolysis of the radioactive substrate [γ32P] ATP; in fact, the synthesis of GSH catalyzed by GshB is coupled to the hydrolysis of ATP. The ATPase activity of rPhGshB required the ...