A homology model for the human theta-class glutathione transferase T1–1

Abstract

A manual threading approach is used to model the human glutathione transferase T1-1 based on the coordinates of the related Theta class enzyme T2-2. The low level of sequence identity (about 20%), found in the C-terminal extension in conjunction with a relative deletion of about five residues makes this a challenging modeling problem. The C-terminal extension contributes to the active site of the molecule and is thus of particular interest for understanding the molecular mechanism of the enzyme. Manual docking of known substrates and non-substrates has implicated potential candidates for the T1-1 catalytic residues involved in the dehalogenation and epoxide-ring opening activities. These include the conserved Theta class residues Arg 107, Trp 115, and the conserved GSTT1 subclass residue His 176. Also, the residue at position 234 is implicated in the modulation of T1-1 activity with different substrates between species.

Structural Basis of the Suppressed Catalytic Activity of Wild-type Human Glutathione Transferase T1-1 Compared to its W234R Mutant

“…16 In that study, five different models (denoted Model 1 to Model 5) were produced with different alignments of the C-terminal parts of the GST T1-1 and GST T2-2 sequences. The alignment of Model 1 was essentially correct except for a few residues.…”

“…15 GST T2-2 shares about 53% amino acid sequence identity with GST T1-1. A GST T1-1 homology model, based on the GST T2-2 structure, has been described earlier 16 but it does not explain the role of residue 234.…”

Gene structure, expression and chromosomal localization of murine Theta class glutathione transferase mGSTT1-1

“…Similarly, mGSTT1-1 exhibited only a low activity towards MS and no activity towards EA, typical substrates of subfamily 2 isoenzymes [5,8,10,13,17]. Crystallographic [43] and homology modelling studies [46] suggest that differences in the volume of the second substrate-binding site (H-site) and differences in C-terminal residue interactions between subfamily 1 and 2 isoenzymes may explain the varied substrate specificities of Theta class GSTs.…”

“…In our laboratory, the successful expression of both recombinant mGSTT1-1 (present study) and hGSTT2-2 [37] with an Nterminal 6iHis tag suggests that the N-terminal domain does not occlude the histidine tag from binding the Ni-NTA resin. In fact, crystallographic studies of hGSTT2 [43] and molecular modelling of hGSTT1 [46] suggest that if a C-terminal histidine tag displaced the C-terminal helix, it might be expected to have a greater influence on the active site than an N-terminal tag.…”

Coping with a Halogenated One-Carbon Diet: Aerobic Dichloromethane-Mineralising Bacteria

“…This difference in mutagenicity beween rat and bacterial enzymes correlates with a previously overlooked thousand-fold difference in affinity for OCM: the rat enzyme has only negligible affinity for OCM (Km -50 mM) compared to bacterial enzymes (Km = 10-60 11M) [46]. Sequence analysis of the GST1) gene in such transconjugants yielded II different sequences encoding GST TI variants, with changes in the protein sequence ranging from single amino acid exchange~ to insertions and deletions of 2 to 32 residues, Sequence variation appeared to cluster ~round the glutathione activation site ( Figure 6) which, in a structural model of GST Tl\ [36], is shielded from the solvent by the C-terminal helix believed to cap the active site of the enzyme [36,37,103]. Sequence analysis of the GST1) gene in such transconjugants yielded II different sequences encoding GST TI variants, with changes in the protein sequence ranging from single amino acid exchange~ to insertions and deletions of 2 to 32 residues, Sequence variation appeared to cluster ~round the glutathione activation site ( Figure 6) which, in a structural model of GST Tl\ [36], is shielded from the solvent by the C-terminal helix believed to cap the active site of the enzyme [36,37,103].…”