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.

[1]  C. Bond,et al.  Structure of a human lysosomal sulfatase. , 1997, Structure.

[2]  R. Read.  Improved Fourier Coefficients for Maps Using Phases from Partial Structures with Errors , 1986 .

[3]  D. Meyer,et al.  Characterization of rat spleen prostaglandin H D-isomerase as a sigma-class GSH transferase. , 1995, The Biochemical journal.

[4]  M. Parker,et al.  A structurally derived consensus pattern for theta class glutathione transferases. , 1996, Protein engineering.

[5]  H. Jörnvall,et al.  Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Hayes,et al.  Characterization of a human class-Theta glutathione S-transferase with activity towards 1-menaphthyl sulphate. , 1992, The Biochemical journal.

[7]  T. Leisinger,et al.  Sequence analysis and expression of the bacterial dichloromethane dehalogenase structural gene, a member of the glutathione S-transferase supergene family , 1990, Journal of bacteriology.

[8]  R. Armstrong.  Glutathione S-transferases: reaction mechanism, structure, and function. , 1991, Chemical research in toxicology.

[9]  C. Tu,et al.  Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli. , 1992, Biochemical and biophysical research communications.

[10]  P. Board,et al.  Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2). , 1996, The Biochemical journal.

[11]  M W Parker,et al.  Evidence for an essential serine residue in the active site of the Theta class glutathione transferases. , 1995, The Biochemical journal.

[12]  G. Kleywegt,et al.  Halloween ... Masks and Bones , 1994 .

[13]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[14]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[15]  G L Gilliland,et al.  Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. , 1995, Biochemistry.

[16]  G J Kleywegt,et al.  Where freedom is given, liberties are taken. , 1995, Structure.

[17]  M. Parker,et al.  Structure and function of glutathione S-transferases. , 1994, Biochimica et biophysica acta.

[18]  B. Ketterer,et al.  The role of glutathione and glutathione transferases in chemical carcinogenesis. , 1990, Critical reviews in biochemistry and molecular biology.

[19]  P. Babbitt,et al.  Exploration of the relationship between tetrachlorohydroquinone dehalogenase and the glutathione S-transferase superfamily. , 1996, Biochemistry.

[20]  G. Chelvanayagam,et al.  Homology model for the human GSTT2 theta class glutathione transferase , 1997, Proteins.

[21]  J. Nash,et al.  Isolation of a mouse theta glutathione S-transferase active with methylene chloride. , 1996, The Biochemical journal.

[22]  M. Parker,et al.  Crystal structure of a theta‐class glutathione transferase. , 1995, The EMBO journal.

[23]  R. Baker,et al.  Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22. , 1995, Genomics.

[24]  M W Parker,et al.  Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 A resolution. , 1992, Journal of molecular biology.

[25]  R. Huber,et al.  Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. , 1996, Journal of molecular biology.

[26]  J. Taylor,et al.  Glutathione S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue. , 1996, The Biochemical journal.

[27]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[28]  J M Thornton,et al.  LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. , 1995, Protein engineering.

[29]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[30]  G J Kleywegt,et al.  Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes. , 1993, Journal of molecular biology.

[31]  S. Kawai,et al.  A bacterial enzyme degrading the model lignin compound β‐etherase is a member of the glutathione‐S‐transferase superfamily , 1993, FEBS letters.

[32]  R. Huber,et al.  X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. , 1994, European journal of biochemistry.

[33]  Conrad C. Huang,et al.  The MIDAS display system , 1988 .

[34]  G L Gilliland,et al.  The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution. , 1992, Biochemistry.

[35]  B. Ketterer,et al.  Theta, a new class of glutathione transferases purified from rat and man. , 1991, The Biochemical journal.

[36]  Axel T. Brunger,et al.  Extension of molecular replacement: a new search strategy based on Patterson correlation refinement , 1990 .

[37]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[38]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[39]  Victoria A. Feher,et al.  Access of ligands to cavities within the core of a protein is rapid , 1996, Nature Structural Biology.

[40]  G Chelvanayagam,et al.  Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues. , 1996, The Biochemical journal.

[41]  F. Quiocho,et al.  Sulphate sequestered in the sulphate-binding protein of Salmonella typhimurium is bound solely by hydrogen bonds , 1985, Nature.

[42]  T A Jones,et al.  Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate. , 1995, Structure.

[43]  H. Bolt,et al.  Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. , 1994, The Biochemical journal.

[44]  J. Martin,et al.  Thioredoxin--a fold for all reasons. , 1995, Structure.

[45]  B. Gillham.  The reaction of aralkyl sulphate esters with glutathione catalysed by rat liver preparations. , 1971, The Biochemical journal.

[46]  B. Gillham.  The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver. , 1973, The Biochemical journal.

[47]  J. Rossjohn,et al.  Preliminary X-ray crystallographic studies of a newly defined human theta-class glutathione transferase. , 1998, Acta crystallographica. Section D, Biological crystallography.

[48]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[49]  H. Okuda,et al.  Novel theta class glutathione S-transferases Yrs-Yrs' and Yrs'-Yrs' in rat liver cytosol: their potent activity toward 5-sulfoxymethylchrysene, a reactive metabolite of the carcinogen 5-hydroxymethylchrysene. , 1994, Biochemical and biophysical research communications.

[50]  D. Eisenberg,et al.  Assessment of protein models with three-dimensional profiles , 1992, Nature.

[51]  B. Mannervik,et al.  Glutathione transferases--structure and catalytic activity. , 1988, CRC critical reviews in biochemistry.