Gene cloning and protein expression of γ-glutamyltranspeptidases from Thermus thermophilus and Deinococcus radiodurans: comparison of molecular and structural properties with mesophilic counterparts

Abstract

γ-Glutamyltranspeptidase (γ-GT) is an ubiquitous enzyme that catalyzes the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. γ-GTs from extremophiles, bacteria adapted to live in hostile environments, were selected as model systems to study the molecular underpinnings of their adaptation to extreme conditions and to find out special properties of potential biotechnological interest. Here, we report the cloning, expression and purification of two members of γ-GT family from two different extremophilic species, Thermus thermophilus (TtGT) and Deinococcus radiodurans (DrGT); the first is an aerobic eubacterium, growing at high temperatures (50-82°C), the second is a polyextremophile, as it tolerates radiations, cold, dehydration, vacuum, and acid. TtGT and DrGT were both synthesized as precursor proteins of 59-60 kDa, undergoing an intramolecular auto-cleavage to yield two subunits of 40 and 19-20 kDa, respectively. However, like the γ-GT from Geobacillus thermodenitrificans, but differently from the other characterized bacterial and eukaryotic γ-GTs, the two new extremophilic enzymes displayed γ-glutamyl hydrolase, but not transpeptidase activity in the 37-50°C temperature range, pH 8.0. The comparison of sequences and structural models of these two proteins with experimental-determined structures of other known mesophilic γ-GTs suggests that the extremophilic members of this protein family have found a common strategy to adapt to different hostile environments. Moreover, a phylogenetic analysis suggests that γ-GTs displaying only γ-glutamyl hydrolase activity could represent the progenitors of the bacterial and eukaryotic counterparts.