Gamma-Glutamylcysteine Production Using Phytochelatin Synthase-Like Enzyme Derived from Nostoc sp. Covalently Immobilized on a Cellulose Carrier.

Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer's disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications.

Reactivity of γ-glutamyl-cysteine with intracellular and extracellular glutathione metabolic enzymes.

Gamma-glutamyl-cysteine (γ-EC) is a precursor of glutathione (GSH) biosynthesis. We investigated whether it functions as a substrate for three intracellular and one extracellular GSH metabolic enzymes, which mediate the antioxidant defence function of GSH. Among them, glutathione peroxidase, glutathione S-transferase and γ-glutamyl transferase (GGT) exhibited substrate specificity for γ-EC, whereas glutathione reductase did not. The specificities of γ-EC and its disulphide form to GGT were comparable to GSH and its oxidized form, GSSG respectively. These results indicate that they can supply GSH constituent amino acids, glutamate, cysteine and cystine through degradation by GGT. γ-EC may contribute valuable antioxidant defence properties as a food and cosmetic additive.

Optimization of Reaction Conditions for γ-Glutamylcysteine Production from Glutathione Using a Phytochelatin Synthase-Like Enzyme from Nostoc sp. Pasteur Culture Collection 7120.

γ-Glutamylcysteine (γ-EC) has antioxidant properties similar to those of glutathione (GSH) and acts as its precursor in mammals. There are a few procedures for the production of γ-EC, such as chemical synthesis or enzymatic synthesis from glutamate and cysteine; however, they are very costly and not suitable for industrial production. A phytochelatin synthase-like enzyme derived from Nostoc sp. Pasteur Culture Collection 7120 (NsPCS) catalyzes the hydrolysis of GSH to γ-EC and glycine in the absence of ATP or other additives. Our research aims to establish an alternative γ-EC production procedure with low cost and high productivity. To this end, we optimized the reaction conditions of NsPCS and characterized its properties in this study. We found that 200 mM potassium phosphate buffer, pH 8.0, at 37 °C, had the highest NsPCS activity among the conditions we tested. Under these conditions, NsPCS had a Km of 385 µM and a Vmax of 26 mol/min/mg-protein. In addition, NsPCS converted 100 mM GSH into γ-EC with high yields. These results suggest that the NsPCS reaction has great potential for the low-cost, industrial-scale production of γ-EC.

Human vault RNA1-1, but not vault RNA2-1, modulates synaptogenesis.

The small non-coding vault RNA (vtRNA) is a component of the vault complex, a ribonucleoprotein complex found in most eukaryotes. vtRNAs regulate a variety of cellular functions when unassociated with the vault complex. Human has four vtRNA paralogs (hvtRNA1-1, hvtRNA1-2, hvtRNA1-3, hvtRNA2-1), which are highly similar and differ only slightly in primary and secondary structure. Despite the increasing research on vtRNAs, a feature that distinguishes one hvtRNA from the others has not been recognized. Recently, we demonstrated that murine vtRNA (mvtRNA) promotes synapse formation by modulating the MAPK signaling pathway. Here we showed that expression ofhvtRNA1-1, but not hvtRNA2-1 increases the expression of synaptic marker proteins, ERK phosphorylation and the number of PSD95 and Synapsin I double positive puncta to an extent similar to that of mvtRNA, suggesting that hvtRNA1-1 may enhance synapse formation. This finding opens new perspectives to uncover the function of the different vtRNA paralogs.