2Department of Life Sciences and Institute of Molecular Biology, National Chung Cheng University, 168 University Road, Minhsiung Township, Chiayi County 62102, Taiwan
3Institute of Genomics and Bioinformatics, National Chung Hsing University, 250 Kuokuang Road, Taichung County 400, Taiwan
* To whom correspondence should be addressed.
Received January 18, 2010; Revision received April 30, 2010
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 ΔG0 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/fo) 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