Summary: | Glutathione (GSH) is a powerful regulator of the physiological redox environment in eukaryotes and prokaryotes. Its antioxidant action, including defence against oxidative damages, detoxification of foreign compounds and toxic metals, preservation of reduced state of protein sulfhydryls, is involved in several cellular pathways. The mechanism of redox homeostasis is mainly based on the intracellular balance between GSH and its oxidised form, GSSG. Biosynthesis of GSH occurs with a mechanism conserved throughout prokaryotes and eukaryotes and involves two sequential steps, both coupled to ATP hydrolysis. The first step, catalysed by g-glutamyl-cysteine ligase (GshA), leads to the formation of g-glutamylcysteine and the second one, producing GSH, is catalysed by glutathione synthetase (GshB). GSH has a more crucial role in microorganisms exposed to oxidative stress conditions, such as the psychrophile Pseudoalteromonas haloplanktis isolated from the Antarctic sea. To characterize the enzyme system for GSH biosynthesis in the first cold adapted microorganism, recombinant forms of GshA and GshB from P. haloplanktis (rPhGshA II and rPhGshB, respectively) were produced and characterized (Albino et al. Mol BioSys 8, 2012, 2405–2414; Biochimie, in press). The investigation covered the study of the substrate specificity of both enzymes, setting up the best ionic and pH conditions for triggering their activities, determination of Km values for all substrates of the catalysed reactions. Both enzymes were already active at 15°C, as required for their cold adaptation. Interestingly and differently from what observed in eukaryotic systems, the reaction rate of rPhGshA II was higher than that reported for rPhGshB, thus suggesting that formation of g-glutamylcysteine was not the rate limiting step of GSH biosynthesis in P. haloplanktis. The inhibitory effect of GSH and GSSG on glutathione synthesis was investigated. Indeed, GSH acted as a non-competitive inhibitor of rPhGshA II and GSSG caused the mono-glutathionylation of the ...
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