Jack U Flanagan

Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site.

BACKGROUND Glutathione S-transferases (GSTs) comprise a multifunctional group of enzymes that play a critical role in the cellular detoxification process. These enzymes reduce the reactivity of toxic compounds by catalyzing their conjugation with glutathione. As a result of their role in detoxification, GSTs have been implicated in the development of cellular resistance to antibiotics, herbicides and clinical drugs and their study is therefore of much interest. In mammals, the cytosolic GSTs can be divided into five distinct classes termed alpha, mu, pi, sigma and theta. The human theta class GST, hGST T2-2, possesses several distinctive features compared to GSTs of other classes, including a long C-terminal extension and a specific sulfatase activity. It was hoped that the determination of the structure of hGST T2-2 may help us to understand more about this unusual class of enzymes. RESULTS Here we present the crystal structures of hGST T2-2 in the apo form and in complex with the substrates glutathione and 1-menaphthyl sulfate. The enzyme adopts the canonical GST fold with a 40-residue C-terminal extension comprising two helices connected by a long loop. The extension completely buries the substrate-binding pocket and occludes most of the glutathione-binding site. The enzyme has a purpose-built novel sulfate-binding site. The crystals were shown to be catalytically active: soaks with 1-menaphthyl sulfate result in the production of the glutathione conjugate and cleavage of the sulfate group. CONCLUSIONS hGST T2-2 shares less than 15% sequence identity with other GST classes, yet adopts a similar three-dimensional fold. The C-terminal extension that blocks the active site is not disordered in either the apo or complexed forms of the enzyme, but nevertheless catalysis occurs in the crystalline state. A narrow tunnel leading from the active site to the surface may provide a pathway for the entry of substrates and the release of products. The results suggest a molecular basis for the unique sulfatase activity of this GST.

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

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. Proteins 33:444–454, 1998.

Identification and characterization of GSTT3, a third murine Theta class glutathione transferase.

A novel Theta class glutathione transferase (GST) isoenzyme from mouse termed mGSTT3 has been identified by analysis of the expressed sequence tag database. The gene encoding mGSTT3 is clustered with the mGSTT1 and mGSTT2 genes on chromosome 10 and has an exon/intron structure that is similar to that of the other Theta class genes. mGSTT3 is expressed strongly in the liver and to a decreasing extent in the kidney and testis. Recombinant mGSTT3-3 expressed in Escherichia coli had a substrate-specificity profile that differed significantly from that of GSTT1-1 and GSTT2-2 isoenzymes. A molecular model of mGSTT3 suggested that, in comparison with GSTT2, a decrease in volume of the hydrophobic substrate-binding site and the loss of the sulphate-binding pocket prevents its use of the GSTT2 substrate 1-menaphthyl sulphate.

Genetic heterogeneity of the structure and function of GSTT2 and GSTP1

In this study new methods for the detection of two polymorphic sites in the GSTP1 coding region have been developed. Both sites are polymorphic in several racial groups and there are significant differences between groups, in the gene frequency at each site. Although previous studies of recombinant GSTP1-1 have suggested that there are significant differences in the specific activity and stability of the I105 or V105 isoforms, no differences in the distribution of GSTP1-1 activities in normal blood donors with different GSTP1 genotypes were detected in this study. These data were obtained with CDNB as a substrate and greater differences may be apparent with different substrates. The structure and organization of the GSTT2 gene was also investigated and a pseudogene that occurs at a polymorphic frequency in European Australians was discovered. This pseudogene can be detected by PCR/RFLP analysis.

Ab initio calculations on hidden modulators of theta class glutathione transferase activity

The glutathione transferases decrease the pKa of glutathione, allowing its deprotonation and the formation of the more reactive thiolate anion. The thiolate is maintained in the active site through a weak conventional hydrogen bond first sphere interaction donated by a Tyr hydroxyl in the Alpha, Mu, Pi, and Sigma glutathione transferase classes that can be modified by other second sphere or indirect thiolate contacts. However, the Theta and Delta class isoforms use a Ser hydroxyl for stabilizing the GSH thiolate, and as such, have a different chemical system compared with that of the Tyr possessed by other classes. We have used high level ab initio methods to investigate this interaction by using a simple methanol methanethiol system as a model. The hydrogen bond strength of this initial first sphere interaction was calculated to be less than that of the Tyr interaction. A putative second sphere interaction exists in the Theta and Delta class structures between Cys or Ser‐14 and Ser‐11 in the mammalian Theta subclass 1 and 2, respectively. The effect of this interaction on the first sphere interaction has also been investigated and found to significantly increase the energy of the bond. Proteins 2000;39:235–243.