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Biochemistry (Moscow)

, Volume 75, Issue 7, pp 919–929 | Cite as

Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis γ-glutamyl transpeptidase

  • Hui-Ping Chang
  • Wan-Chi Liang
  • Rui-Cin Lyu
  • Meng-Chun Chi
  • Tzu-Fan Wang
  • Kuo-Liang Su
  • Hui-Chih Hung
  • Long-Liu LinEmail author
Article

Abstract

The role of the C-terminal region of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581–585, 577–585, 576–585, 566–585, 558–585, 523–585, and 479–585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-γ-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Δ(576–585), pro-Δ(566–585), and pro-Δ(558–585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Δ(577–585), Δ(576–585), Δ(566–585), Δ(558–585), Δ(523–585), and Δ(479–585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and ΔG 0 for the unfolding. BlGGT-4aa and BlGGT sedimented at ∼4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/f o) of BlGGT-4aa, BlGGT, Δ(581–585), and Δ(577–585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.

Key words

Bacillus licheniformis γ-glutamyl transpeptidase C-terminal truncation autocatalytic processing analytical ultracentrifugation 

Abbreviations

AEW

average emission wavelength

BlGGT, EcGGT, and HpGGT

Bacillus licheniformis, Escherichia coli, and Helicobacter pylori γ-glutamyl transpeptidases

CD

circular dichroism

IPTG

isopropyl-β-D-thiogalactopyranoside

L-γ-Glu-p-NA

L-γ-glutamyl-p-nitroanilide

Ntn

N-terminal nucleophile

p-NA

p-nitroaniline

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • Hui-Ping Chang
    • 1
  • Wan-Chi Liang
    • 1
  • Rui-Cin Lyu
    • 1
  • Meng-Chun Chi
    • 1
  • Tzu-Fan Wang
    • 2
  • Kuo-Liang Su
    • 3
  • Hui-Chih Hung
    • 3
  • Long-Liu Lin
    • 1
    Email author
  1. 1.Department of Applied ChemistryNational Chiayi UniversityChiayi CountyTaiwan
  2. 2.Department of Life Sciences and Institute of Molecular BiologyNational Chung Cheng UniversityChiayi CountyTaiwan
  3. 3.Institute of Genomics and BioinformaticsNational Chung Hsing UniversityTaichung CountyTaiwan

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