Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis

The oligomeric states of Bacillus licheniformis and Escherichia coli gamma-glutamyltranspeptidases (BIGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BIGGT has a sedimentation coefficient of 5.12 5, which can be transformed into an experimental molecular mass of approximately 62,680 Da. The monomeric conformation is conserved in EcGGT. SDS-PAGE analysis and cross-linking studies further proved that the autocatalytically processed BIGGT 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. BIGGT and EcGGT had a T(m) value of 59.5 and 49.2 degrees C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Chemical unfolding of BIGGT 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 6 M GdnHCl. BIGGT was active in the presence of 4 M 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. (C) 2011 Elsevier B.V. All rights reserved.