title: The maturation mechanism of γ-glutamyl transpeptidases: Insights from the crystal structure of a precursor mimic of the enzyme from Bacillus licheniformis and from site-directed mutagenesis studies.
abstract: γ-Glutamyl transpeptidases (γ-GTs) are members of N-terminal nucleophile hydrolase superfamily. They are synthetized as single-chain precursors, which are then cleaved to form mature enzymes. Basic aspects of autocatalytic processing of these pro-enzymes are still unknown. Here we describe the X-ray structure of the precursor mimic of Bacillus licheniformis γ-GT (BlGT), obtained by mutating catalytically important threonine to alanine (T399A-BlGT), and report results of autoprocessing of mutants of His401, Thr415, Thr417, Glu419 and Arg571. Data suggest that Thr417 is in a competent position to activate the catalytic threonine (Thr399) for nucleophilic attack of the scissile peptide bond and that Thr415 plays a major role in assisting the process. On the basis of these new structural results, a possible mechanism of autoprocessing is proposed. This mechanism, which guesses the existence of a six-membered transition state involving one carbonyl and two hydroxyl groups, is in agreement with all the available experimental data collected on γ-GTs from different species and with our new Ala-scanning mutagenesis data.
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia, Naples I-80126, Italy; Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone, 16, Naples 80133, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pica
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, 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 60004, Taiwan.
authors.forename: Yi-Yu
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia, Naples I-80126, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: d'Ischia
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia, Naples I-80126, Italy; Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone, 16, Naples 80133, Italy. Electronic address: antonello.merlino@unina.it.
authors.forename: Antonello
authors.initials: A
authors.lastname: Merlino
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D018360:Crystallography, X-Ray; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics
chemical_list: D005723:gamma-Glutamyltransferase; D000409:Alanine
keywords: Autoprocessing; Single-point mutation; X-ray crystallography; γ-GT precursor; γ-Glutamyltranspeptidase