International Journal of Biological Macromolecules 48 (2011) 414–422 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Biophysical characterization of Bacillus licheniformis and Escherichia coli -glutamyltranspeptidases: A comparative analysis Jia-Ci Yang a,1 , Wan-Chi Liang a,1 , Yi-Yu Chen a , Meng-Chun Chi a , Huei-Fen Lo b , Hsiang-Ling Chen b , Long-Liu Lin a,∗ a Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan b Department of Food Science and Technology, Hungkuang University, Shalu, Taichung County 433, Taiwan article info Article history: Received 2 November 2010 Received in revised form 22 December 2010 Accepted 4 January 2011 Available online 14 January 2011 Keywords: -Glutamyltranspeptidase Oligomeric state Analytical ultracentrifuge Thermal unfolding Chemical denaturation abstract The oligomeric states of Bacillus licheniformis and Escherichia coli -glutamyltranspeptidases (BlGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BlGGT has a sedimentation coefficient of 5.12 S, which can be transformed into an experimental molec- ular 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 BlGGT and EcGGT form a heterodimeric association. Unfolding analyses using circular dichroism and tryptophan emis- sion 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 6 M GdnHCl. BlGGT was active in the pres- ence 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction -Glutamyltranspeptidase (GGT; 2.3.2.2) is a member of the N- terminal nucleophile (Ntn) hydrolase superfamily [1] and catalyzes the first step in glutathione degradation [2,3]. The inactive precur- sor of all the known Ntn-hydrolases undergoes an intramolecular autoprocessing to generate the mature and catalytically active enzyme. A conserved threonine residue in GGTs, Thr391 in Escherichia coli GGT (EcGGT) [4] and Thr380 in Helicobacter pylori GGT (HpGGT) [5], serves as the N-terminal nucleophile and is required for both maturation and catalytic activity. The typical fold of Ntn-hydrolases consists of a four-layered catalytically active -core structure [6]. This core is formed by two antiparallel - sheets packed against each other, and these -sheets are covered by a layer of antiparallel -helices on one side [1,7]. Recently, three- dimensional structures of EcGGT and HpGGT have been solved [5,8]. The structure of acyl-enzyme intermediate of EcGGT con- ∗ Corresponding author. Tel.: +886 5 2717969; fax: +886 5 2717901. E-mail address: llin@mail.ncyu.edu.tw (L.-L. Lin). 1 These authors contributed equally to this work. firms the functional role of Thr391 and the location of the donor substrate-binding site [8]. Further studies on these two enzymes have provided the structural evidence for autocatalytic processing [9,10] and critical roles of the C-terminal region in autoprocessing and catalysis [11]. The open reading frame of EcGGT codes a polypeptide precursor consisting of a signal peptide of 25 amino acid residues, a large sub- unit (L-subunit) of 363 residues, and a small subunit (S-subunit) of 190 residues (Swiss-Prot P18956). Similarly, Bacillus licheniformis GGT (BlGGT) is synthesized as a 61.259-kDa polypeptide precur- sor made up a signal peptide of 25 residues, a L-subunit of 374 residues, and a S-subunit of 187 residues (Swiss-Prot Q65KZ6). Earlier, the gene encoding either BlGGT or EcGGT was cloned and over-expressed in recombinant E. coli cells [12,13]. Deletion anal- ysis of both enzymes showed that removal of the signal peptide significantly affects their functional expressions in E. coli [14,15]. To the best of our knowledge, there has been no report dealing with the biophysical characterization of both enzymes, and it is unknown the oligomeric state of these two proteins in solution. In the present study, we compared the biophysical properties of BlGGT and EcGGT using analytical ultracentrifugation (AUC), cir- cular dichroism (CD), and intrinsic tryptophan fluorescence. The 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.01.006