Exploring the unfolding mechanism of γ-glutamyltranspeptidases: the case of the thermophilic enzyme from Geobacillus thermodenitrificans.
γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze 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. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.
Department of Chemical Sciences, University of Naples, Naples, Italy.
Andrea
A
Pica
Irene
I
Russo Krauss
Immacolata
I
Castellano
Mosè
M
Rossi
Francesco
F
La Cara
Giuseppe
G
Graziano
Filomena
F
Sica
Antonello
A
Merlino
D019943:Amino Acid Substitution; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability; D005456:Fluorescent Dyes / Q000737:chemistry; D056506:Geobacillus / Q000201:enzymology*; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence; D044366:Transition Temperature; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D005723:gamma-Glutamyltransferase; D019791:Guanidine