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Structural basis of the suppressed catalytic activity of wild-type human glutathione transferase T1-1 compared to its W234R mutant
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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2006 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 355, no 1, 96-105 p.Article in journal (Refereed) Published
Abstract [en]

The crystal structures of wild-type human theta class glutathione-S-transferase (GST) T1-1 and its W234R mutant, where Trp234 was replaced by Arg, were solved both in the presence and absence of S-hexyl-glutathione. The W234R mutant was of interest due to its previously observed enhanced catalytic activity compared to the wild-type enzyme. GST T1-1 from rat and mouse naturally contain Arg in position 234, with correspondingly high catalytic efficiency. The overall structure of GST T1-1 is similar to that of GST T2-2, as expected from their 53% sequence identity at the protein level. Wild-type GST T1-1 has the side-chain of Trp234 occupying a significant portion of the active site. This bulky residue prevents efficient binding of both glutathione and hydrophobic substrates through steric hindrance. The wild-type GST T1-1 crystal structure, obtained from co-crystallization experiments with glutathione and its derivatives, showed no electron density for the glutathione ligand. However, the structure of GST T1-1 mutant W234R showed clear electron density for S-hexyl-glutathione after co-crystallization. In contrast to Trp234 in the wild-type structure, the side-chain of Arg234 in the mutant does not occupy any part of the substrate-binding site. Instead, Arg234 is pointing in a different direction and, in addition, interacts with the carboxylate group of glutathione. These findings explain our earlier observation that the W234R mutant has a markedly improved catalytic activity with most substrates tested to date compared to the wild-type enzyme. GST T1-1 catalyzes detoxication reactions as well as reactions that result in toxic products, and our findings therefore suggest that humans have gained an evolutionary advantage by a partially disabled active site.

Place, publisher, year, edition, pages
2006. Vol. 355, no 1, 96-105 p.
Keyword [en]
glutathione transferase, T1-1, protein X-ray crystallography, protein–ligand interaction, Binding Sites/genetics, Catalysis, Crystallography; X-Ray, Glutathione/analogs & derivatives/chemistry, Glutathione Transferase/*chemistry/genetics/metabolism, Humans, Mutation; Missense, Protein Conformation, Research Support; Non-U.S. Gov't
National Category
Structural Biology Biochemistry and Molecular Biology
URN: urn:nbn:se:uu:diva-76733DOI: 10.1016/j.jmb.2005.10.049PubMedID: 16298388OAI: oai:DiVA.org:uu-76733DiVA: diva2:104645
Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2010-03-02Bibliographically approved
In thesis
1. Design of Glutathione Transferase Variants for Novel Activities with Alternative Substrates
Open this publication in new window or tab >>Design of Glutathione Transferase Variants for Novel Activities with Alternative Substrates
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) play a pivotal role in cellular defense, since they are main contributors to the inactivation of genotoxic compounds of exogenous and endogenous origins. Directed evolution was used to improve the catalytic activities of Theta class GST T1-1 toward different substrates. The library was constructed by recombination of cDNA coding for human GST T1-1 and rodent Theta class GSTs, resulting in the F2-F5 generations. The clones were heterologously expressed in Escherichia coli and screened for variants with enhanced alkyltransferase activity. A mutant, F2:1215, with a 70-fold increased catalytic efficiency with 4-nitrophenethyl bromide (NPB) compared to human GST T1-1, was isolated from the second generation. NPB was used as a surrogate substrate of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in order to facilitate the screening process. The catalytic efficiency of the F2:1215 with BCNU had improved 170-fold compared to wild-type human GST T1-1, suggesting that NPB is a suitable model substrate for the anticancer drug BCNU. The sequence of the F2:1215 mutant differs from wild-type human GST T1-1 by three residues; one of these differences is Arg234, which corresponds to Trp in the human enzyme. Upon replacing the Trp234 in the human GST T1-1 with Arg, the resulting mutant (hTrp234Arg) showed enhanced alkyltransferase activity with a wide range of substrates (e.g. haloalkanes and other typical GSTs substrates). The three-dimensional structures of both wild-type human GST T1-1 and hTrp234Arg mutant help to explain the higher activity showed by of hTrp234Arg mutant compared to wild-type enzyme. The reciprocal mutation of the residue 234 in mouse GST T1-1 to that found in human, mArg234Trp, caused a dramatic decrease in the activity of the mouse enzyme to be similar to human GST T1-1. This indicates that residue 234 can be considered as a master switch of activities between human and rodent GST T1-1. Another important residue in the C-terminal helix of GST T1-1 is Met232. Although residue 232 points away from the H-site, it influences the catalytic activity and substrate selectivity of the mouse GST T1-1. A minor modification of Met232 induces major changes in the substrate-activity profile of the mouse GST T1-1 to favor novel substrates such as isothiocyanates and hydroperoxides and decreases the activity toward substrates that catalyzed by the wild-type enzyme.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 77 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 720
National Category
Biochemistry and Molecular Biology Organic Chemistry
Research subject
Biochemistry; Organic Chemistry
urn:nbn:se:uu:diva-119768 (URN)978-91-554-7733-2 (ISBN)
Public defence
2010-04-12, C10:305, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2010-03-22 Created: 2010-03-01 Last updated: 2010-03-22Bibliographically approved

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