The first X-Ray structure of a cold-adapted glutathione synthetase

Glutathione (GSH) is the major antioxidant molecule in most living organisms. Besides its protective role in contrasting oxidative free radical species and in the removal of toxic metals, GSH is involved in redox homeostasis and cell signaling; furthermore, it is essential to maintain cellular proteins in their reduced state and acts as a post-translational regulator of protein function. The synthesis of GSH from its constituent amino acids involves two ATP-requiring enzymatic steps catalyzed by glutamate cysteine ligase (GshA) and glutathione synthetase (GshB), respectively. Its biosynthetic route has been described in details for a variety of organisms; however, nothing is known about this process in psychrophilic microorganisms. In particular, although various GshBs have been purified and sequenced, none of these enzymes belong to an organism living at low temperatures. Very recently, a recombinant form of GshB from the cold-adapted bacterium Pseudoalteromonas haloplanktis (PhGshB) has been purified. The activity of PhGshB was assayed through a direct method, measuring the release of inorganic phosphate from radiolabelled ATP. PhGshB is active at 10°C and its activity significantly increases with temperature, at least up to 30°C; the Km for ATP ranges between 0.14 and 0.25 mM in the 10 – 30°C interval. The crystal structure of PhGshB has been determined and refined at 2.3 Ǻ resolution. Structural information is combined with a characterization of the thermal stability of the enzyme performed by circular dichroism and fluorescence measurements. A comparison between our data and those obtained for the closely related GshB from the mesophilic Escherichia coli [1-3], sharing 69% identity with PhGshB, is presented in the attempt to understand the mechanisms of cold-adaptation of the psychrophilic enzyme.