Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis gamma-glutamyl transpeptidase.
The role of the C-terminal region of Bacillus licheniformis gamma-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-gamma-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Delta(576-585), pro-Delta(566-585), and pro-Delta(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 Delta(577-585), Delta(576-585), Delta(566-585), Delta(558-585), Delta(523-585), and Delta(479-585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and DeltaG(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, Delta(581-585), and Delta(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.
Chang
Hui-Ping
HP
Department of Applied Chemistry, National Chiayi University, Taiwan.
Liang
Wan-Chi
WC
Lyu
Rui-Cin
RC
Chi
Meng-Chun
MC
Wang
Tzu-Fan
TF
Su
Kuo-Liang
KL
Hung
Hui-Chih
HC
Lin
Long-Liu
LL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D019281:Dimerization; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D017384:Sequence Deletion; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005723:gamma-Glutamyltransferase