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  • 1. Akoachere, Monique
    et al.
    Iozef, Rimma
    Rahlfs, Stefan
    Deponte, Marcel
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Creighton, Donald J
    Schirmer, Heiner
    Becker, Katja
    Characterization of the glyoxalases of the malarial parasite Plasmodium falciparum and comparison with their human counterparts.2005In: Biol Chem, ISSN 1431-6730, Vol. 386, no 1, p. 41-52Article in journal (Refereed)
    Abstract [en]

    The glyoxalase system consisting of glyoxalase I (GloI) and glyoxalase II (GloII) constitutes a glutathione-dependent intracellular pathway converting toxic 2-oxoaldehydes, such as methylglyoxal, to the corresponding 2-hydroxyacids. Here we describe a complete glyoxalase system in the malarial parasite Plasmodium falciparum. The biochemical, kinetic and structural properties of cytosolic GloI (cGloI) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) were directly compared with the respective isofunctional host enzymes. cGloI and cGloII exhibit lower K(m) values and higher catalytic efficiencies (k(cat)/K(m) ) than the human counterparts, pointing to the importance of the system in malarial parasites. A Tyr185Phe mutant of cGloII shows a 2.5-fold increase in K(m) , proving the contribution of Tyr185 to substrate binding. Molecular models suggest very similar active sites/metal binding sites of parasite and host cell enzymes. However, a fourth protein, which has highest similarities to GloI, was found to be unique for malarial parasites; it is likely to act in the apicoplast, and has as yet undefined substrate specificity. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were active in the lower nanomolar range. The Glo system of Plasmodium will be further evaluated as a target for the development of antimalarial drugs.

  • 2.
    Andersson, Malena
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Exploring protein evolution by saturation mutagenesis of the GST M2-2 active site residue 2102005In: FEBS Journal, Vol. 272, p. 81 Suppl-Article in journal (Other scientific)
  • 3. Dourado, Daniel F. A. R.
    et al.
    Fernandes, Pedro Alexandrino
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ramos, Maria Joao
    Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3: A Computational Study Identifies a Dual Role for Glutathione2014In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 118, no 31, p. 5790-5800Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Delta 5-androstene-3,17-dione (Delta(5)-AD) and Delta(5)-pregnene-3,20-dione (Delta(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Delta(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Delta(5)-AD C4 atom to the Delta(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal.mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the 03 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leul11, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Delta(4)-AD crystal structure and is consistent with all available experimental data.

  • 4.
    Dourado, Daniel F. A. R.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, p. 8069-8078Article in journal (Refereed)
    Abstract [en]

    Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

  • 5. Edalat, Maryam H
    et al.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Peptide phage display for probing GST-protein interactions.2005In: Methods Enzymol, ISSN 0076-6879, Vol. 401, p. 354-67Article in journal (Refereed)
    Abstract [en]

    Phage display is a powerful strategy for identifying protein-peptide interactions. Glutathione transferases (GSTs) play prominent roles in the cellular protection against oxidative stress by catalyzing detoxication reactions. In addition, GSTs seem to act in signaling pathways by means of interaction with other macromolecules such as protein kinases. This chapter describes how the technique of peptide phage display can be used to identify possible partners in GST-protein complexes.

  • 6.
    Edalat, Maryam
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Technology, Department of Materials Science. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Axelsson, Lars-Göran
    Selective expression of detoxifying glutathione transferases in mouse colon: effect of experimental colitis and the presence of bacteria.2004In: Histochem Cell Biol, ISSN 0948-6143, Vol. 122, no 2, p. 151-9Article in journal (Refereed)
  • 7.
    Eklund, Birgitta I.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs2007In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 8, p. 1098-1103Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the β-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

  • 8.
    Emrén, Lars O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kurtovic, Sanela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Runarsdottir, Arna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Larsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Functionally diverging molecular quasi-species evolve by crossing two enzymes2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 29, p. 10866-10870Article in journal (Refereed)
    Abstract [en]

    Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.

  • 9.
    Grahn, Elin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Novotny, Marian
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Jakobsson, Emma
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Gustafsson, Ann
    Grehn, Leif
    Olin, Birgit
    Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Madsen, Dennis
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Wahlberg, Mårten
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, Bengt
    Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kleywegt, Gerard J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    New crystal structures of human glutathione transferase A1-1 shed light on glutathione binding and the conformation of the C-terminal helix.2006In: Acta Crystallogr D Biol Crystallogr, ISSN 0907-4449, Vol. 62, no Pt 2, p. 197-207Article in journal (Refereed)
    Abstract [en]

    Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

  • 10.
    Gustafsson, Ann
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Pettersson, Pär L.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Grehn, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-12001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 51, p. 15835-15845Article in journal (Refereed)
    Abstract [en]

    The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.

  • 11. Hederos, Sofia
    et al.
    Broo, Kerstin
    Jakobsson, Emma
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kleywegt, Gerard J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, Bengt
    Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Baltzer, Lars
    Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    A new enzyme by rational design - the incorporation of a single His residue enables efficient thioester hydrolysis by human glutathione transferase A1-12004In: Proc. Nat. Acad. Sci., Vol. 101, p. 13163-13167Article in journal (Refereed)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 12. Hederos, Sofia
    et al.
    Broo, Kerstin S
    Jakobsson, Emma
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. ICM.
    Kleywegt, Gerard J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. ICM.
    Mannervik, Bengt
    Department of Biochemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Baltzer, Lars
    Department of Chemistry. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Incorporation of a single His residue by rational design enables thiol-ester hydrolysis by human glutathione transferase A1-1.2004In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 101, no 36, p. 13163-7Article in journal (Refereed)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 13.
    Hegazy, Usama M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits2006In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 13, no 9, p. 929-936Article in journal (Refereed)
    Abstract [en]

    Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.

  • 14.
    Hegazy, Usama M
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Stenberg, Gun
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Functional role of the lock and key motif at the subunit interface of glutathione transferase P1-12004In: Journal of Biological Chemistry, Vol. 279, p. 9586-9596Article in journal (Refereed)
  • 15.
    Hegazy, Usama M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Tars, Kaspars
    Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 590, SE-751 24 Uppsala, Sweden.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Modulating Catalytic Activity by Unnatural Amino Acid Residues in a GSH-Binding Loop of GST P1-12008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 3, p. 811-826Article in journal (Refereed)
    Abstract [en]

    The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.

  • 16.
    Hegazy, Usama
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Site-directed substitution of the key residue at the subunit interface of GST P1-1 by S-alkylcysteine residues sustains glutathione binding and catalytic activity of the enzyme2005In: FEBS Journal, Vol. 272, p. 87 Suppl-Article in journal (Refereed)
  • 17. Ito, Mika
    et al.
    Shibata, Aya
    Zhang, Jie
    Hiroshima, Michio
    Sako, Yasushi
    Nakano, Yukiko
    Kojima-Aikawa, Kyoko
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Shuto, Satoshi
    Ito, Yoshihiro
    Morgenstern, Ralf
    Abe, Hiroshi
    Universal Caging Group for the in-Cell Detection of Glutathione Transferase Applied to 19F NMR and Bioluminogenic Probes2012In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 10, p. 1428-1432Article in journal (Refereed)
  • 18.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Combinatorial protein chemistry in three dimensions: a paradigm for the evolution of glutathione transferases with novel activities2006In: Toxicology of Glutathione Transferases / [ed] Awasthi, Yogesh C., CRC Taylor & Francis, Boca Raton , 2006, p. 47-69Chapter in book (Other academic)
  • 19.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Exploring the importance of an active site residue on the stereo and regio selectivity of Mu class glutathione transferases2005In: FEBS Journal, Vol. 272, p. 88 Suppl-Article in journal (Refereed)
  • 20.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Regio- and enantioselectivities in epoxide conjugations are modulated by residue 210 in Mu class glutathione transferases.2005In: Protein Eng Des Sel, ISSN 1741-0126, Vol. 18, no 12, p. 607-16Article in journal (Refereed)
    Abstract [en]

    The homologous human glutathione transferases (GSTs) M1-1 and M2-2 have similar catalytic activities with many electrophilic substrates, but differ strikingly in their conjugation of epoxides with glutathione. Residue 210, Thr in GST M2-2 and Ser in GST M1-1, is a key active-site component in determining the activity profile with epoxide substrates. This residue is hypervariable in Mu class GSTs, suggesting that it has special significance in the evolution of new functions. The present study shows that minor modifications of this residue can have major consequences for the enzyme-catalyzed epoxide conjugations. In general, a Ser at position 210 gives the highest catalytic efficiency, but the relatively high activity with an Ala placed on this position demonstrates that a hydroxyl group is not required. In contrast, a Thr residue suppresses the activity with epoxides by several orders of magnitude without major effects on the activity with alternative GST substrates. Residue 210 influences both the regio- and enantioselectivity with chiral and prochiral epoxides of stilbene and styrene and influences the distribution of isomeric glutathione conjugates. Thus, residue 210 contributes to both stereoselective recognition of the substrates and to partitioning of the isomeric reactants to the alternative transition states leading to separate chiral products.

  • 21.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Norrgård, Malena A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications2007In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 9, p. 1374-1381Article in journal (Refereed)
    Abstract [en]

    Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.

  • 22.
    Ivarsson, Ylva
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Norrgård, Malena A
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Tars, Kaspar
    Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    A positively selected residue influences enzyme functionalities2006In: The FASEB Journal, Vol. 20, p. A474-Article, review/survey (Other (popular scientific, debate etc.))
  • 23.
    Johansson, Ann-Sofie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-32002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 19, p. 16648-16654Article in journal (Refereed)
    Abstract [en]

    Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.

  • 24.
    Johansson, Ann-Sofie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 1051998In: Journal of Molecular Cell Biology, ISSN 1674-2788, E-ISSN 1759-4685, Vol. 278, no 3, p. 687-698Article in journal (Refereed)
    Abstract [en]

    Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

  • 25. Josephy, David P
    et al.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry.
    Molecular Toxicology2006Book (Refereed)
  • 26. Josephy, P. David
    et al.
    Taylor, Patricia L.
    Vervaet, Gaby
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Screening and characterization of variant Theta-class glutathione transferases catalyzing the activation of ethylene dibromide to a mutagen2006In: Environmental and Molecular Mutagenesis, ISSN 0893-6692, E-ISSN 1098-2280, Vol. 47, no 9, p. 657-665Article in journal (Refereed)
    Abstract [en]

    Ethylene dibromide (EDB) is a widespread environmental pollutant and mutagen/carcinogen. Certain Theta-class glutathione transferases (GSTs), enzymes that catalyze the reaction of reduced glutathione (GSH) with electrophiles, activate EDB to a mutagen. Previous studies have shown that human GST T1-1, but not rat GST T2-2, activates EDB. We have constructed an E. coli lacZ reversion mutagenicity assay system in which expression of recombinant GST supports activation of EDB to a mutagen. Hexa-histidine N-terminal tagging of GST T1-1 results in greatly enhanced expression of the recombinant enzyme and gives a lacZ strain that shows a mutagenic response to EDB at extremely low levels ( approximately 1 ng EDB per plate). The hexa-histidine-tagged enzyme was purified in one step by Ni(2+)-affinity chromatography. We applied the lacZ mutagenicity assay to the rapid screening of a library of variant GST Theta enzymes. Sequence variants with altered catalytic activities were identified, purified, and characterized.

     

  • 27.
    Kjellander, Marcus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mazari, Aslam M.A.
    Department of Neurochemistry, Stockholm University, SE-10691, Sweden.
    Boman, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Inorganic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening and stability2014In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, p. 59-63Article in journal (Refereed)
    Abstract [en]

    The previously uncharacterized Drosophila melanogaster Epsilon class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ∊-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that for enzymes in solution and no leakage of enzyme from the matrix could be observed.

  • 28.
    Kurtovic, Sanela
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Emrén, Lars O
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Runarsdottír, Arna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Larsson, Anna-Karin
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Identification of functionally diverging quasi-species in molecular enzyme evolution2006In: The FASEB Journal, Vol. 20, p. A470-Article, review/survey (Other (popular scientific, debate etc.))
  • 29.
    Kurtovic, Sanela
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Runarsdottir, Arna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Emrén, Lars O.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Larsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library2007In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 5, p. 243-256Article in journal (Refereed)
    Abstract [en]

    A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space. For an incisive analysis, the rows of the data matrix, corresponding to the different enzyme variants, were individually scaled to unit length, thus accounting for different expression levels of the enzymes. The columns representing the activities with alternative substrates were subsequently individually normalized to unit variance and a zero mean. By this standardization, the data were adjusted to comparable orders of magnitude. Three molecular quasi-species were recognized by multivariate K-means and principal component analyses. Two of them encompassed the parental GST M1-1 and GST M2-2. A third one diverged functionally by displaying enhanced activities with some substrates and suppressed activities with signature substrates for GST M1-1 and GST M2-2. A fourth cluster contained mutants with impaired functions and was not regarded as a quasi-species. Sequence analysis of representatives of the mutant clusters demonstrated that the majority of the variants in the diverging novel quasi-species were structurally similar to the M1-like GSTs, but distinguished themselves from GST M1-1 by a Ser to Thr substitution in the active site. The data show that multivariate analysis of functional profiles can identify small structural changes influencing the evolution of enzymes with novel substrate-activity profiles.

  • 30.
    Kurtovic, Sanela
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Runarsdottir, Arna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Larsson, Anna-Karin
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Emrén, Lars O
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Diverging substrate specificites from a glutathione transferase library analyzed by multivariate methods2005In: FEBS Journal, Vol. 272, p. 90 Suppl-Article in journal (Refereed)
  • 31.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Five Decades with Glutathione and the GSTome2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 9, p. 6072-6083Article in journal (Other academic)
    Abstract [en]

    Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept "molecular quasi-species," we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution.

  • 32.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Optimizing the heterologous expression of glutathione transferase2005In: Methods of Enzymology, Vol. 401, p. 254-265Article in journal (Refereed)
    Abstract [en]

    The heterologous expression of a protein may be enhanced by silent mutations in the coding region of its corresponding DNA. This simple approach has been successfully used for optimized production of a number of glutathione-linked enzymes. For example, the yield of human glutathione transferase M2-2 was elevated by 140-fold in a clone isolated by immunoscreening of a library of plasmids with randomized synonymous codons in the 5'-segment of the region encoding the enzyme.

  • 33.
    Mannervik, Bengt
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Board, Philip G
    Hayes, John D
    Listowsky, Irving
    Pearson, William R
    Nomenclature for mammalian soluble glutathione transferases2005In: Methods of Enzymology, Vol. 401, p. 1-8Article in journal (Refereed)
    Abstract [en]

    The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs.

  • 34.
    Modén, Olof
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Zhang, Wei
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine2012In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 25, no 4, p. 189-197Article in journal (Refereed)
    Abstract [en]

    Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structureactivity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with approximate to 10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93 library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.

  • 35. Mukanganyama, Stanley
    et al.
    Widersten, Mikael
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Naik, Yogeshkumar S.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Hasler, Julia A.
    Inhibition of glutathione S-transferases by antimalarial drugs: Possible implications for circumventing anticancer drug resistance2002In: Int J Cancer, Vol. 97, p. 700-705Article in journal (Refereed)
  • 36. Musdal, Yaman
    et al.
    Hegazy, Usama M.
    Aksoy, Yasemin
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-12013In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 205, no 1, p. 53-62Article in journal (Refereed)
    Abstract [en]

    Objective: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. Methods: We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. Results: We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. Conclusion and practical implications: In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

  • 37.
    Nilsson, Lisa O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Edalat, Maryam
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Pettersson, Pär L.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Aromatic residues in the C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism2002In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1598, no 1-2, p. 199-205Article in journal (Refereed)
    Abstract [en]

    Human glutathione transferase A1-1 (GST A1-1) has a flexible C-terminal segment that forms a helix (alpha9) closing the active site upon binding of glutathione and a small electrophilic substrate such as 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of active-site ligands, the C-terminal segment is not fixed in one position and is not detectable in the crystal structure. A key residue in the alpha9-helix is Phe 220, which can interact with both the enzyme-bound glutathione and the second substrate, and possibly guide the reactants into the transition state. Mutation of Phe 220 into Ala and Thr was shown to reduce the catalytic efficiency of GST A1-1. The mutation of an additional residue, Phe 222, caused further decrease in activity. The presence of a viscosogen in the reaction medium decreased the kinetic parameters k(cat) and k(cat)/K(m) for the conjugation of CDNB catalyzed by wild-type GST A1-1, in agreement with the view that product release is rate limiting for the substrate-saturated enzyme. The mutations cause a decrease of the viscosity dependence of both kinetic parameters, indicating that the motion of the alpha9-helix is linked to catalysis in wild-type GST A1-1. The isomerization reaction with the alternative substrate Delta(5)-androstene-3,17-dione (AD) is affected in a similar manner by the viscosogens. The transition state energy of the isomerization reaction, like that of the CDNB conjugation, is lowered by Phe 220 as indicated by the effects of the mutations on k(cat)/K(m). The results demonstrate that Phe 220 and Phe 222, in the dynamic C-terminal segment, influence rate-determining steps in the catalytic mechanism of both the substitution and the isomerization reactions.

  • 38.
    Norrgård, Malena A
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Tars, Kaspars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 13, p. 4876-4881Article in journal (Refereed)
    Abstract [en]

    All molecular species in an organism are connected physically and functionally to other molecules. In evolving systems, it is not obvious to what extent functional properties of a protein can change to selective advantage and leave intact favorable traits previously acquired. This uncertainty has particular significance in the evolution of novel pathways for detoxication, because an organism challenged with new xenobiotics in the environment may still require biotransformation of previously encountered toxins. Positive selection has been proposed as an evolutionary mechanism for facile adaptive responses of proteins to changing conditions. Here, we show, by saturation mutagenesis, that mutations of a hypervariable residue in human glutathione transferase M2-2 can differentially change the enzyme's substrate-activity profile with alternative substrates and, furthermore, enable or disable dissimilar chemical reactions. Crystal structures demonstrate that activity with epoxides is enabled through removal of steric hindrance from a methyl group, whereas activities with an orthoquinone and a nitroso donor are maintained in the variant enzymes. Given the diversity of cellular activities in which a single protein can be engaged, the selective transmutation of functional properties has general significance in molecular evolution.

  • 39.
    Norrgård, Malena A
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-SiteResidue Sustaining Catalytic Promiscuity2011In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 412, no 1, p. 111-120Article in journal (Refereed)
    Abstract [en]

    The substrate-binding H-site of human glutathionetransferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 mu mol min(-1) mg(-1) resulted primarily from an increased k(cat) value (70 s(-1)). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 mu mol min(-1) mg(-1). In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conservedIle10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrateselectivity profiles in GSTs by a few mutations. 

  • 40. Park, Hee-Sung
    et al.
    Nam, Sung-Hun
    Lee, Jin Kak
    Yoon, Chang No
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Photochemistry and Molecular Science. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Benkovic, Stephen J
    Kim, Hak-Sung
    Design and evolution of new catalytic activity with an existing protein scaffold.2006In: Science, ISSN 1095-9203, Vol. 311, no 5760, p. 535-8Article in journal (Refereed)
    Abstract [en]

    The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alphabeta/betaalpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat/Km)app of 1.8 x 10(2) (mole/liter)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.

  • 41.
    Pettersson, Pär L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Johansson, Ann-Sofie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Transmutation of Human Glutathione Transferase A2-2 with Peroxidase Activity into an Efficient Steroid Isomerase2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, p. 30019-30022Article in journal (Refereed)
    Abstract [en]

    A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

  • 42.
    Pettersson, Pär L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    The role of glutathione in the isomerization of Delta(5)-androstene-3,17-dione catalyzed by human glutathione transferase A1-12001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 15, p. 11698-11704Article in journal (Refereed)
    Abstract [en]

    Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.

  • 43.
    Raffalli-Mathieu, Francoise
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry. Department of Biochemistry and Organic Chemistry, Biochemistry.
    Human glutathione transferase A3-3 active as steroid double-bond isomerase2005In: Methods of Enzymology, Vol. 401, p. 265-278Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs) constitute a superfamily of detoxifying enzymes with a major role in protecting cellular macromolecules from reactive electrophilic compounds. A growing body of evidence suggests, however, that at least certain glutathione transferases are involved in other essential cellular processes, such as cellular signaling and anabolic pathways. One of them is the human GST A3-3, which is selectively expressed in steroidogenic organs and which efficiently catalyzes the obligatory isomerization of the Delta(5)-ketosteroid precursors in the biosynthesis of progesterone and testosterone. In this chapter, we summarize the current knowledge on human GST A3-3 and describe methods for heterologous expression and functional characterization of the enzyme.

  • 44.
    Ridderstrom, M
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry.
    Jemth, P
    Cameron, AD
    Mannervik, B
    The active-site residue Tyr-175 in human glyoxalase II contributes to binding of glutathione derivatives2000In: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, ISSN 0167-4838, Vol. 1481, no 2, p. 344-348Article in journal (Refereed)
    Abstract [en]

    Tyrosine-175 located in the active site of human glyoxalase II was replaced by phenylalanine in order to study the contribution of this residue to catalysis. The mutation had a marginal effect on the k(cat) value determined using S-D-lactoylglutathione as

  • 45. Seeley, Stacy K
    et al.
    Poposki, Julie A
    Maksimchuk, John
    Tebbe, Jill
    Gaudreau, Jon
    Mannervik, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry.
    Bull, Arthur W
    Metabolism of oxidized linoleic acid by glutathione transferases: peroxidase activity toward 13-hydroperoxyoctadecadienoic acid.2006In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1760, no 7, p. 1064-70Article in journal (Refereed)
    Abstract [en]

    The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.

  • 46. Shibata, Aya
    et al.
    Nakano, Yukiko
    Ito, Mika
    Araki, Mika
    Zhang, Jie
    Yoshida, Yasuhiko
    Shuto, Satoshi
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mogenstern, Ralf
    Ito, Yoshihiro
    Abe, Hiroshi
    Fluorogenic probes using 4-substituted-2-nitrobenzenesulfonyl derivatives as caging groups for the analysis of human glutathione transferase catalyzed reactions2013In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 138, no 24, p. 7326-7330Article in journal (Refereed)
    Abstract [en]

    We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the sigma value in the same way as the non-enzymatic reaction, whereas the dependence of the sigma value of the GST mu and pi was not as pronounced as that of GST alpha.

  • 47.
    Shokeer, Abeer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Larsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Residue 234 in glutathione transferase T1-1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates2005In: Abstracts of the 30th FEBS Congress: FEBS journal Special issue (Vol. 272, Issue s1), 2005, Vol. 272, p. 99 Suppl-Conference paper (Refereed)
  • 48.
    Shokeer, Abeer
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Larsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Residue 234 in glutathione transferase T1-1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates2005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 388, no Pt 1, p. 387-92Article in journal (Refereed)
    Abstract [en]

    GST (glutathione transferase) T1-1 plays an important role in the biotransformation of halogenated alkanes, which are used in large quantities as solvents and occur as environmental pollutants. Many reactions that are catalysed by GST T1-1 qualify as detoxification processes, but some reactions with dihalogenated alkanes lead to reactive products more toxic than the substrates. Murine GST T1-1 is particularly active with dichloromethane, which may explain the high carcinogenicity of dichloromethane in the mouse. Human GST T1-1 activity is considerably lower with halogenated hydrocarbons and some related substrates. Human GST T1-1 is polymorphic with a frequent null phenotype, suggesting that it is advantageous, under some circumstances, to lack the functional enzyme, which catalyses GSH conjugations that may cause bioactivation. The present study shows that amino acid residue 234 is a determinant of the differences in catalytic efficiency between the human and the rodent enzymes. The replacement of Trp234 in human GST T1-1 by arginine, found in the rodent enzyme, enhanced the alkyltransferase activity by an order of magnitude with a series of homologous iodoalkanes and some typical GST substrates. The specific activity of the alternative mutant Trp234-->Lys was lower than for the parental human GST T1-1 with many substrates, showing that a positive charge is not sufficient for increased activity. The enhanced activity of Trp234-->Arg with alkylating agents was dependent on the substrate tested, whereas no increase of the peroxidase activity with cumene hydroperoxide was noted. Residue 234 therefore is also involved in the control of the substrate selectivity of GST T1-1.

  • 49.
    Sinning, I
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kleywegt, G J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Cowan, S W
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Reinemer, P
    Dirr, H W
    Huber, R
    Gilliland, G L
    Armstrong, R N
    Ji, X
    Board, P G
    Olin, B
    Chemistry, Department of Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, B
    Chemistry, Department of Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Jones, T A
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes.1993In: J Mol Biol, ISSN 0022-2836, Vol. 232, no 1, p. 192-212Article in journal (Refereed)
    Abstract [en]

    The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.

  • 50.
    Sinning, I
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Kleywegt, G J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, B
    Department of Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Board, P G
    Jones, T A
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    The active site in class alpha glutathione transferases1993In: Structure and Function of Glutathione Transferases, CRC Press, Boca Raton, FL , 1993, p. 75-85Chapter in book (Other scientific)
12 1 - 50 of 63
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