Albino, Antonella (2011) The glutathione biosynthesis in the psychrophile Pseudoalteromonas haloplanktis. [Tesi di dottorato] (Unpublished)
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|Item Type:||Tesi di dottorato|
|Uncontrolled Keywords:||Glutathione biosynthesis; Psychrophile; Oxidative stress|
|Date Deposited:||06 Dec 2011 15:51|
|Last Modified:||30 Apr 2014 19:48|
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 presence of the other two substrates, glycine and γ-glutamylcysteine (γ-Glu-Cys). rPhGshB showed its maximum activity in the 7.4-8.2 pH range. The enzyme activity required also the presence of a divalent cation and at 5 mM Mg++ reached its maximum. The kinetic parameters of rPhGshB at 15°C, the optimum value for growth of P. haloplanktis, were determined. The enzyme showed a comparable affinity for ATP and γ-Glu-Cys (Km = 0.26 mM and 0.25 mM, respectively), whereas a lower affinity was determined for glycine (Km = 0.75 mM). The comparison of these data with those of the corresponding enzyme from other sources showed the remarkable similarity with Escherichia coli; a similar lower affinity for glycine compared to the other two substrates was found in the other sources. Enzyme inhibition studies showed that GSSG is an inhibitor of rPhGshB. The effect of temperature on the kinetic parameters of rPhGshB was analysed in the temperature range 10-30°C. The data showed that rPhGshB is already active at 10°C and its Vmax significantly increased with temperature up to 25°C; on the other hand, in the temperature interval considered, a minimum variation of Km was observed. The kcat values were then used to draw an Arrhenius plot and in the range of linearity (10-25°C) an activation energy of 75 kJ/mol was determined, a value quite high for a psychrophilic enzyme. The thermostability of rPhGshB was analyzed using a thermal inactivation profile and an extrapolated half-life of 10 min at 50.5°C was derived. This value was slightly high for a psychrophilic enzyme, although not unusual for enzymes involved in the control of the cellular redox state. A value of 208 kJ/mol was calculated for the energy of activation of the heat inactivation process; this value was intermediate between those usually obtained for psychrophilic and mesophilic enzymes, respectively. Finally, the development of a new crystallization technique allowed the obtainment of crystal forms of rPhGshB, useful for the determination of the three-dimensional structure of the enzyme by X-ray diffraction. The biochemical characterization of rPhGshA-II was started, using the same assay adopted for rPhGshB. Preliminary data obtained showed that the enzyme activity of rPhGshA-II requires a reducing agent in the reaction mixture; furthermore, the presence of the other two substrates glutamate and cysteine, is also required. The activity of rPhGshA-II needed a higher concentration of Mg++ (10-20 mM) compared to that already determined for rPhGshB. When the characterization of the molecular and biochemical properties of PhGshA-II will be completed, it will be possible to reconstitute in vitro for the first time the system for the biosynthesis of glutathione in a psychrophilic organism.
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