Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis.
The oligomeric states of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases (BlGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BlGGT has a sedimentation coefficient of 5.12S, which can be transformed into an experimental molecular mass of approximately 62,680Da. The monomeric conformation is conserved in EcGGT. SDS-PAGE analysis and cross-linking studies further proved that the autocatalytically processed BlGGT and EcGGT form a heterodimeric association. Unfolding analyses using circular dichroism and tryptophan emission fluorescence revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and EcGGT had a T(m) value of 59.5 and 49.2°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Chemical unfolding of BlGGT was independent to the pH value ranging from 5 to 10, whereas the pH environment was found to significantly influence the GdnHCl-induced denaturation of EcGGT. Both enzymes did not reactivate from the completely unfolded states, accessible at 6M GdnHCl. BlGGT was active in the presence of 4M NaCl, whereas the activity of EcGGT was significantly decreased at the high-salt condition. Taken together, these findings suggest that the biophysical properties of the homologous GGTs from two mesophilic sources are quite different.
Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan.
Jia-Ci
JC
Yang
Wan-Chi
WC
Liang
Yi-Yu
YY
Chen
Meng-Chun
MC
Chi
Huei-Fen
HF
Lo
Hsiang-Ling
HL
Chen
Long-Liu
LL
Lin
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D058767:Protein Unfolding / Q000187:drug effects; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D012965:Sodium Chloride; D005723:gamma-Glutamyltransferase; D019791:Guanidine