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
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
D'ISCHIA, MARCO;
2016
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 dataI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.