Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1864, Issue 2, Pages 195-203Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2015.10.006
Keywords
gamma-Glutamyltranspeptidase; gamma-GT precursor; Autoprocessing; Single-point mutation; X-ray crystallography
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Funding
- National Science Council of Taiwan [NSC-100-2313-B-415-003-MY3]
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gamma-Glutamyl transpeptidases (gamma-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 gamma-GT (BIGT), obtained by mutating catalytically important threonine to alanine (T399A-BIGT), 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 gamma-GTs from different species and with our new Ala-scanning mutagenesis data. (C) 2015 Elsevier B.V. All rights reserved.
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