γ-Glutamyl transpeptidase architecture: Effect of extra sequence deletion on autoprocessing, structure and stability of the protein from Bacillus licheniformis

gamma-Glutamyl transpeptidases (gamma-GTs, EC 23.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (gamma-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its gamma-glutamyl group to water (hydrolysis), amino acids or small peptides (transpeptidation). These proteins utilize a conserved Thr residue to process their chains into a large and a small subunit that then form the catalytically competent enzyme. Multiple sequence alignments have shown that some bacterial gamma-GTs, including that from Bacillus licheniformis (BlGT), possess an extra sequence at the C-terminal tail of the large subunit, whose role is unknown. Here, autoprocessing, structure, catalytic activity and stability against both temperature and the chemical denaturant guanidinium hydrochloride of six BlGT extra-sequence deletion mutants have been characterized by SDS-PAGE, circular dichroism, intrinsic fluorescence and homology modeling. Data suggest that the extra sequence has a crucial role in enzyme activation and structural stability. Our results assist in the development of a structure-based interpretation of the autoprocessing reaction of gamma-GTs and are helpful to unveil the molecular bases of their structural stability. (C) 2014 Elsevier B.V. All rights reserved.