Glutathione biosynthesis in a cold-adapted source

Glutathione, the most abundant non-enzymatic cellular thiol, regulates the redox environment through a balance of its reduced (GSH) and oxidised (GSSG) forms. Its antioxidant tasks include the defence against oxidative damages caused by reactive oxygen species (ROS), detoxification from toxic compounds, protection from oxidation of protein sulfhydryls. Hence, this tripeptide affects many cellular functions, such as transport, metabolism, cell cycle, carcinogenesis, apoptosis; furthermore, it is involved in the post-translational regulation of protein functions through the S-glutathionylation of specific cysteine residues. The enzyme mechanism for GSH biosynthesis is conserved throughout prokaryotes and eukaryotes and includes two sequential steps for the assemblage of glutamate, cysteine and glycine into GSH, the driving force being guaranteed by the hydrolysis of ATP. In most prokaryotes, the two reaction steps are catalysed by two distinct enzymes, ?-glutamyl-cysteine ligase (GshA) and glutathione synthetase (GshB). In this work, the GSH biosynthesis was investigated for the first time in a cold-adapted microorganism, such as Pseudoalteromonas haloplanktis. This psychrophile, perfectly adapted to ROS protection, exhibits intriguing features in the functional role and biosynthesis of GSH. First, some antioxidant enzymes from this source are S-glutathionylated; second, differently from other prokaryotes containing a single GshA and GshB, P. haloplanktis has two redundant GshA gene products (GshA I and GshA II) besides one GshB. In this study a detailed characterization of the recombinant forms of these enzymes was realised, focusing on the different role played by the redundant GshAs. The cold adaptations of the three enzymes of P. haloplanktis were compared and two distinct complete systems for GSH synthesis were reconstituted and analysed in order to dissect partial and complete reaction rates for GSH biosynthesis, even for potential biotechnological applications.