title: γ-Glutamyl transpeptidase architecture: Effect of extra sequence deletion on autoprocessing, structure and stability of the protein from Bacillus licheniformis.
abstract: γ-Glutamyl transpeptidases (γ-GTs, EC 2.3.2.2) are a class of ubiquitous enzymes which initiate the cleavage of extracellular glutathione (γ-Glu-Cys-Gly, GSH) into its constituent glutamate, cysteine, and glycine and catalyze the transfer of its γ-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 γ-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 γ-GTs and are helpful to unveil the molecular bases of their structural stability.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan.
authors.forename: Yi-Hui
authors.initials: YH
authors.lastname: Lo
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan.
authors.forename: Yi-Yu
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan. Electronic address: llin@mail.ncyu.edu.tw.
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy; Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy. Electronic address: antonello.merlino@unina.it.
authors.forename: Antonello
authors.initials: A
authors.lastname: Merlino
keywords: Deletion mutants; Ntn hydrolase; Protein stability; Site-directed mutagenesis; γ-Glutamyl transpeptidase