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  • 1.
    Aslam, Muhammad
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dahlberg, Olle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Transgenic Overexpression of Glutathione Transferase E7 in Drosophila Attenuates Toxicity of Organic Isothiocyanates Affecting Survival and OvipositionManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract.  Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significant extension in life-span when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies showed no significant effect in the first few days, and subsequently showed a somewhat lower survival rate. At 1 mM AITC concentration, no toxicity was noted. However, the oviposition activity was dramatically enhanced from a very low level in wild-type flies reared in the presence of 1 mM AITC to values an order of magnitude higher for the transgenic flies. The results demonstrate a clear protective effect of GSTE7 against exposure to ITC allelochemicals which can affect both life-span and fecundity of female flies.

  • 2. Bocedi, Alessio
    et al.
    Fabrini, Raffaele
    Lo Bello, Mario
    Caccuri, Anna Maria
    Federici, Giorgio
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cornish-Bowden, Athel
    Ricci, Giorgio
    Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function2016Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, nr 52, s. 26739-26749Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of n(H) are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.

  • 3. Bolelli, K.
    et al.
    Musdal, Yaman
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Aki-Yalcin, E.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Yalcin, I.
    Synthesis and activity mechanism of some novel 2-substituted benzothiazoles as hGSTP1-1 enzyme inhibitors2017Inngår i: SAR and QSAR in environmental research (Print), ISSN 1062-936X, E-ISSN 1029-046X, Vol. 28, nr 11, s. 927-940Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human GSTP1-1 is one of the most important proteins, which overexpresses in a large number of human tumours and is involved in the development of resistance to several anticancer drugs. So, it has become an important target in cancer treatment. In this study, 12 benzothiazole derivatives were synthesized and screened for their in vitro inhibitory activity for hGSTP1-1. Among these compounds, two of them (compounds #2 and #5) have been found to be the leads when compared with the reference drug etoposide. In order to analyse the structure-activity relationships (SARs) and to investigate the binding side interactions of the observed lead compounds, a HipHop pharmacophore model was generated and the molecular docking studies were performed by using CDocker method. In conclusion, it is observed that the lead compounds #2 and #5 possessed inhibitory activity on the hGSTP1-1 by binding to the H-site as a substrate in which the para position of the phenyl ring of the benzamide moiety on the benzothiazole ring is important. Substitution at this position with a hydrophobic group that reduces the electron density at the phenyl ring is required for the interaction with the H side active residue Tyr108.

  • 4. Brunnström, Åsa
    et al.
    Hamberg, Mats
    Griffiths, William J.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Claesson, Hans-Erik
    Biosynthesis of 14,15-hepoxilins in human L1236 hodgkin lymphoma cells and eosinophils2011Inngår i: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 46, nr 1, s. 69-79Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA(3) 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB(3) 13 (R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-D). Incubation of L1236 cells with synthetic 14,15-HxA(3) 11(S) also led to the formation of 14,15-HxA(3)-C and 14,15-HxA(3)-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA(3) to 14,15-HxA(3)-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.

  • 5. Cebula, Marcus
    et al.
    Turan, Ilke Simsek
    Sjödin, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Thulasingam, Madhuranayaki
    Brock, Joseph
    Chmyrov, Volodymyr
    Widengren, Jerker
    Abe, Hiroshi
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Haeggström, Jesper Z.
    Rinaldo-Matthis, Agnes
    Akkaya, Engin U.
    Morgenstern, Ralf
    Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase2016Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 38316Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

  • 6. Cuevas, Carlos
    et al.
    Huenchuguala, Sandro
    Munoz, Patricia
    Villa, Monica
    Paris, Irmgard
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Segura-Aguilar, Juan
    Glutathione Transferase-M2-2 Secreted from Glioblastoma Cell Protects SH-SY5Y Cells from Aminochrome Neurotoxicity2015Inngår i: Neurotoxicity research, ISSN 1029-8428, E-ISSN 1476-3524, Vol. 27, nr 3, s. 217-228Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    U373MG cells are able to take up aminochrome that induces glutathione transferase M2-2 (GSTM2) expression in a concentration-dependent manner where 100 A mu M aminochrome increases GSTM2 expression by 2.1-fold (P < 0.001) at 3 h. The uptake of H-3-aminochrome into U373MG cells was significantly reduced in the presence of 2 A mu M nomifensine (P < 0.001) 100 A mu M imipramine (P < 0.001) and 50 mM dopamine (P < 0.001). Interestingly, U373MG cells excrete GSTM2 into the conditioned medium and the excretion was significantly increased (2.7-fold; P < 0.001) when the cells were pretreated with 50 A mu M aminochrome for 3 h. The U373MG-conditioned medium containing GSTM2 protects SH-SY5Y cells incubated with 10 A mu M aminochrome. The significant protection provided by U373MG-conditioned medium in SH-SY5Y cells incubated with aminochrome was dependent on GSTM2 internalization into SH-SY5Y cells as evidenced by (i) uptake of C-14-GSTM2 released from U373MG cells into SH-SY5Y cells, a process inhibited by anti-GSTM2 antiserum; (ii) lack of protection of U373MG-conditioned medium in the presence of anti-GSTM2 antiserum on SH-SY5Y cells treated with aminochrome; and (iii) lack of protection of conditioned medium from U373MGsiGST6 that expresses an siRNA directed against GSTM2 on SH-SY5Y cells treated with aminochrome. In conclusion, our results demonstrated that U373MG cells protect SH-SY5Y cells against aminochrome neurotoxicity by releasing GSTM2 into the conditioned medium and subsequent internalization of GSTM2 into SH-SY5Y cells. These results suggest a new mechanism of protection of dopaminergic neurons mediated by astrocytes by releasing GSTM2 into the intersynaptic space and subsequent internalization into dopaminergic neuron in order to protect these cells against aminochrome neurotoxicity.

  • 7. Dourado, Daniel F. A. R.
    et al.
    Fernandes, Pedro Alexandrino
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    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 Glutathione2014Inngår i: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 118, nr 31, s. 5790-5800Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 8.
    Dourado, Daniel F. A. R.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 45, s. 8069-8078Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 9. Ertan-Bolelli, Tugba
    et al.
    Bolelli, Kayhan
    Musdal, Yaman
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Yildiz, Ilkay
    Aki-Yalcin, Esin
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Yalcin, Ismail
    Design and synthesis of 2-substituted-5-(4-trifluoromethylphenyl-sulphonamido)benzoxazole derivatives as human GST P1-1 inhibitors2018Inngår i: Artificial Cells, Nanomedicine, and Biotechnology, ISSN 2169-1401, Vol. 46, nr 3, s. 510-517Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The glutathione transferases (GSTs) are a family of widely distributed Phase II detoxification enzymes. GST P1-1 is frequently overexpressed in rat and human tumours. It is suggested that overexpression of hGST P1-1 by human tumor cells may play a role in resistance to cancer chemotherapy. Hence, hGST P1-1 can be a promising target for cancer treatment. In this study, new hGST P1-1 inhibitors, 2-(4-substitutedphenyl/benzyl)-5-(4-trifluoromethylphenylsulphonamido) benzoxazole derivatives (Va-Vk) have been designed and synthesized. Surprisingly, in vitro hGST P1-1 enzyme inhibition studies demonstrated that all of the tested compounds except Vj had better activity than the reference drug EA and it is also correlated with the docking results. Additionally we compared the interactions with hGST P1-1 enzyme of newly synthesized compound Vh (bearing CF3 group) and previously synthesized compound 5f (bearing NO2 group). According to the docking results, compound Vh bound to the hGST P1-1 enzyme with a higher affinity compared to 5f. Therefore, we can consider that these data make a sense and can explain its higher activity. The compounds that obtained from this research could be used as scaffolds in design of new potent hGST P1-1 inhibitors useful in the treatment of the resistance of cancer chemotherapy.

  • 10. Fedulova, Natalia
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Experimental conditions affecting functional comparison of highly active glutathione transferases2011Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, nr 1, s. 16-23Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. The combination of time-dependent and enzyme concentration-dependent loss of activity and the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

  • 11. Fedulova, Natalia
    et al.
    Raffalli-Mathieu, Françoise
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of porcine Alpha-class glutathione transferase A1-12011Inngår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 507, nr 2, s. 205-211Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissues and animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence of our clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to our cloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1(∗). The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactions was at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

  • 12. Govindarajan, Sridhar
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Silverman, Joshua A.
    Wright, Kathy
    Regitsky, Drew
    Hegazy, Usama
    Purcell, Thomas J.
    Welch, Mark
    Minshull, Jeremy
    Gustafsson, Claes
    Mapping of Amino Acid Substitutions Conferring Herbicide Resistance in Wheat Glutathione Transferase2015Inngår i: ACS Synthetic Biology, ISSN 2161-5063, Vol. 4, nr 3, s. 221-227Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have used design of experiments (DOE) and systematic variance to efficiently explore glutathione transferase substrate specificities caused by amino acid substitutions. Amino acid substitutions selected using phylogenetic analysis were synthetically combined using a DOE design to create an information-rich set of gene variants, termed infologs. We used machine learning to identify and quantify protein sequence-function relationships against 14 different substrates The resulting models were quantitative and predictive, serving as a guide for engineering of glutathione transferase activity toward a diverse set of herbicides Predictive quantitative models like those presented here have broad applicability for bioengineering.

  • 13.
    Hegazy, Usama M.
    et al.
    National Research Centre (NRC), Egypt.
    Musdal, Yaman
    Hacettepe University, Turkey.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hidden Allostery in Human Glutathione Transferase P1-1 Unveiled by Unnatural Amino Acid Substitutions and Inhibition Studies2013Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, nr 9, s. 1509-1514Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kat and 300-fold increased K-M(GSH) values, displays an apparent Hill coefficient of 0.82 +/- 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency k(cat)/K-M(GSH) to near the wild-type value but still yields Hill coefficients of 0.61 +/- 0.08 and 0.86 +/- 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 +/- 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

  • 14. Honaker, Matthew T.
    et al.
    Acchione, Mauro
    Zhang, Wei
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Atkins, William M.
    Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites2013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 25, s. 18599-18611Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning induced fit versus conformational selection has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1-1 (GSTA1-1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1-1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that local molten globule behavior optimizes detoxication enzymes.

  • 15. Huenchuguala, Sandro
    et al.
    Munoz, Patricia
    Zavala, Patricio
    Villa, Monica
    Cuevas, Carlos
    Ahumada, Ulises
    Graumann, Rebecca
    Nore, Beston F.
    Couve, Eduardo
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Paris, Irmgard
    Segura-Aguilar, Juan
    Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction2014Inngår i: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 10, nr 4, s. 618-630Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit H-3-dopamine uptake, which is inhibited by 2 mu M of nomifensine and 15 mu M of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 mu M purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 mu M aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A(1), and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A(1) pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/-tubulin (tubulin, ) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.

  • 16. Huenchuguala, Sandro
    et al.
    Sjödin, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Segura-Aguilar, Juan
    Novel Alpha-Synuclein Oligomers Formed with the Aminochrome-Glutathione Conjugate Are Not Neurotoxic2019Inngår i: Neurotoxicity research, ISSN 1029-8428, E-ISSN 1476-3524, Vol. 35, nr 2, s. 432-440Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aminochrome induces neurotoxic alpha-synuclein oligomer formation relevant to the etiology of Parkinson's disease. Oxidative stress produces aminochrome from dopamine, but conjugation with glutathione catalyzed by glutathione transferase M2-2 significantly decreases aminochrome-induced toxicity and alpha-synuclein oligomer formation. Notably, in the presence of the aminochrome-glutathione conjugate, previously unknown species of alpha-synuclein oligomers are formed. These aminochrome-glutathione oligomers of alpha-synuclein differ from formerly characterized oligomers and (i) have high molecular weight, and are stable and SDS-resistant, as determined by the Western blot method, (ii) show positive NBT-quinone-protein staining, which indicates the formation of alpha-synuclein adducts containing aminochrome. Furthermore, aminochrome-glutathione alpha-synuclein oligomers (iii) have distinctive shape and size, as determined by transmission electron microscopy, and (iv) are not toxic in U373MG cells. In conclusion, glutathione conjugated with aminochrome induces a new type of alpha-synuclein oligomers of a different size and shape, which have no demonstrable toxicity.

  • 17. Ito, Mika
    et al.
    Shibata, Aya
    Zhang, Jie
    Hiroshima, Michio
    Sako, Yasushi
    Nakano, Yukiko
    Kojima-Aikawa, Kyoko
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    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 Probes2012Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, nr 10, s. 1428-1432Artikkel i tidsskrift (Fagfellevurdert)
  • 18. Josephy, P. David
    et al.
    Pan, Dan
    Ianni, Michael D.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Functional studies of single-nucleotide polymorphic variants of human glutathione transferase T1-1 involving residues in the dimer interface2011Inngår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 513, nr 2, s. 87-93Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferase T1-1 catalyses detoxication and bioactivation processes in which glutathione conjugates are formed from endogenous and xenobiotic substrates, including alkylating agents and halogenated alkanes. Although the common null polymorphism of the human GSTT1 gene has been studied extensively, little is known about the consequences of GSTT1 single-nucleotide polymorphisms (SNPs). Here, we have examined the effects of two SNPs that alter amino acid residues in the dimer interface of the GST T1-1 protein and one that causes a conservative substitution in the core of the subunit. Variant proteins were expressed in an Escherichia coli strain in which the metabolism of ethylene dibromide to a glutathione conjugate leads to lacZ reversion mutations. We measured the kinetic properties of the enzymes with the characteristic substrate 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and determined the specific activities with several other substrates. Circular dichroism spectroscopy was used to measure protein thermal denaturation profiles. Variant T104P, which has been reported as inactive, showed weak but detectable activity with each substrate. Variant R76S was expressed at lower levels and showed much-reduced thermal stability. The results are interpreted in the context of the three-dimensional structure of human GST T1-1.

  • 19. Kjellander, Marcus
    et al.
    Mazari, Aslam M. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Boman, Mats
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Johansson, Gunnar
    Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability2014Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 446, s. 59-63Artikkel i tidsskrift (Fagfellevurdert)
    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 c-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 of enzymes in solution, and no leakage of enzyme from the matrix could be observed.

  • 20. Larsson, Emilia
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Raffalli-Mathieu, Françoise
    Quantitative and selective polymerase chain reaction analysis of highly similar human alpha-class glutathione transferases2011Inngår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 412, nr 1, s. 96-101Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Alpha-class glutathione transferases (GSTs) found expressed in human tissues constitute a family of four homologous enzymes with contrasting enzyme activities. In particular, GST A3-3 has been shown to contribute to the biosynthesis of steroid hormones in human cells and is selectively expressed in steroidogenic tissues. The more ubiquitous GST A1-1, GST A2-2, and GST A4-4 appear to be primarily involved in detoxification processes and are expressed at higher levels than GST A3-3. We are interested in studying the cell and tissue expression of the GST A3-3 gene, yet the existence of highly expressed sequence-similar homologs and of several splice variants is a serious challenge for the specific detection of unique transcript species. We found that published polymerase chain reaction (PCR) primers for GST A3-3 lack the specificity required for reliable quantitative analysis. Therefore, we designed quantitative PCR (qPCR) primers with greatly increased discrimination power for the human GSTA3 full-length transcript. The improved primers allow accurate discrimination between GST A3-3 and the other alpha-class GSTs and so are of great value to studies of the expression of the GSTA3 gene. The novel primers were used to quantify GSTA3 transcripts in human embryonic liver and steroidogenic cell lines.

  • 21.
    Lindström, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mazari, Aslam M. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Musdal, Yaman
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Inhibition characteristics of equine steroid isomerase EcaGST A3-3Manuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxifying enzymes found in all organisms. However it is also the most efficient steroid double-bond isomerase known in mammals. In contrast to the rodents, Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes it a more suitable model for human steroidogenesis than the murine one. Inhibition of EcaGST A3-3 might help treat reproductive and neurodegenerative disorders. We screened an FDA-approved library of 1040 compounds for the ability as novel inhibitors of EcaGST A3-3. Our results revealed anthralin, sennoside A, tannic acid and ethacrynic acid as the most potent, submicromolar-range inhibitors of EcaGST A3-3 with the natural substrate Δ5-androstene-3,17-dione.

  • 22.
    Lindström, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mazari, Aslam M. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Musdal, Yaman
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Potent inhibitors of equine steroid isomerase EcaGST A3-32019Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, nr 3, artikkel-id e0214160Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 mu M concentration afforded at least 50% inhibition of EcaGST A3-3. The most potent inhibitors, anthralin, sennoside A, tannic acid, and ethacrynic acid, were characterized by IC50 values in the submicromolar range when assayed with the natural substrate Delta(5)-androstene-3,17-dione.

  • 23.
    Lindström, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Peer, Shawna M.
    Ing, Nancy H.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of equine GST A3-3 as a steroid isomerase2018Inngår i: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 178, s. 117-126Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ5-androstene-3,17-dione to Δ4-androstene-3,17-dione and of Δ5-pregnene-3,20-dione to Δ4-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse (Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones.

  • 24.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Five Decades with Glutathione and the GSTome2012Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 9, s. 6072-6083Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 25.
    Mannervik, Bengt
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sjödin, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Blood-Brain Barrier-Penetrating 6-Halogenopurines Suitable as Pro-Probes for Positron Emission Tomography are Substrates for Human Glutathione Transferases2016Inngår i: Pharmaceutical Bioprocessing, ISSN 2048-9145, Vol. 4, nr 2, s. 25-30Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    6-Chloro- and 6-bromopurines can cross the blood-brain barrier and in situ give rise to substrates of multidrug resistance-associated proteins (MRPs). The electrophilic purines form glutathione conjugates in reactions catalyzed by intracellular glutathione transferases (GSTs), and the conjugates are subsequently exported from the cells by ATP-dependent membrane transporters. In rodent model systems it has been demonstrated that suitably radiolabeled 6-halogenopurines by this scheme are pro-probes useful in monitoring the functionality of MRPs in intact brains using positron emission tomography. Prior to applications in human subjects it is imperative to establish the purine pro-probes as effective substrates for human GSTs occurring in brain and other tissues. We have developed a spectrophotometric assay for the glutathione conjugation and determined specific activities with a range of human GSTs as well as some rat GSTs for comparison. The ubiquitous GST P1-1 showed the highest activities with the 6-halogenopurines, which bodes well for the application of pro-probes for human investigations.

  • 26.
    Mazari, Aslam M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dahlberg, Olle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Overexpression of Glutathione Transferase E7 in Drosophila Differentially Impacts Toxicity of Organic Isothiocyanates in Males and Females2014Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 10, artikkel-id e110103Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract. Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significantly increased survival when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies show a significantly lower survival rate. Oviposition activity was enhanced in transgenic flies. The effect was significant in transgenic females reared in the absence of ITCs as well as in the presence of 0.15 mM PEITC or 1 mM AITC. Thus the GSTE7 transgene elicits responses to exposure to ITC allelochemicals which differentially affect life-span and fecundity of male and female flies.

  • 27.
    Mazari, Aslam M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hegazy, Usama M.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Identification of new inhibitors for human hematopoietic prostaglandin D-2 synthase among FDA-approved drugs and other compounds2015Inngår i: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 229, s. 91-99Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Objective: Hematopoietic prostaglandin D-2 synthase (HPGDS) is a member of the Sigma class glutathione transferases (GSTs) catalyzing the isomerization of prostaglandin H-2 to prostaglandin D-2, a mediator of allergy and inflammation responses. Selective inhibitors of human HPGDS are expected to be of therapeutic importance in relieving symptoms related to allergy and asthma. Hence, a collection of diverse FDA-approved compounds was screened for potential novel applications as inhibitors of HPGDS. Methods: The catalytic activity of purified HPGDS was used for inhibition studies in vitro. Results: Our inhibition studies revealed 23 compounds as effective inhibitors of HPGDS with IC50 values in the low micromolar range. Erythrosine sodium, suramin, tannic acid and sanguinarine sulfate were characterized with IC50 values of 0.2, 0.3, 0.4, and 0.6 mu M, respectively. Kinetic inhibition analysis showed that erythrosine sodium is a nonlinear competitive inhibitor of HPGDS, while suramin, tannic acid and sanguinarine sulfate are linear competitive inhibitors. Conclusion: The results show that certain FDA-approved compounds may have pharmacological effects not previously realized that warrant further consideration in their clinical use.

  • 28.
    Mazari, Aslam M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene2016Inngår i: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 5, s. 141-145Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) and the related 2,4-dinitrotoluene (DNT) aretoxic environmental pollutants. The biotransformation and detoxication of these persistent compoundsin higher organisms are of great significance from a health perspective as well as for the biotechnological challenge of bioremediation of contaminated soil. We demonstrate that different human glutathionetransferases (GSTs) and GSTs from the fruit fly Drosophila melanogaster are catalysts of the biotransformationof TNT and DNT. The human GSTs had significant but modest catalytic activities with both DNT and TNT. However, D. melanogaster GSTE6 and GSTE7 displayed outstanding high activities withboth substrates.

  • 29. Moden, Olof
    et al.
    Zhang, Wei
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine2012Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 25, nr 4, s. 189-197Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 30. Modén, Olof
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Glutathione Transferases in the Bioactivation of Azathioprine2014Inngår i: Redox and Cancer Part A, San Diego: Elsevier, 2014, s. 199-244Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adverse side effects. The prodrug is activated by glutathione conjugation and release of 6-mercaptopurine, a reaction most efficiently catalyzed by glutathione transferase (GST) A2-2. Among five genotypes of GST A2-2, the variant A2*E has threefold fourfold higher catalytic efficiency with azathioprine, suggesting that the expression of A2*E could boost 6-mercaptopurine release and adverse side effects in treated patients. Structure-activity studies of the GST A2-2 variants and homologous alpha class GSTs were made to delineate the determinants of high catalytic efficiency compared to other alpha class GSTs. Engineered chimeras identified GST peptide segments of importance, and replacing the corresponding regions in low-activity GSTs by these short segments produced chimeras with higher azathioprine activity. By contrast, H-site mutagenesis led to decreased azathioprine activity when active-site positions 208 and 213 in these favored segments were mutagenized. Alternative substitutions indicated that hydrophobic residues were favored. A pertinent question is whether variant A2*E represents the highest azathioprine activity achievable within the GST structural framework. This issue was addressed by mutagenesis of H-site residues assumed to interact with the substrate based on molecular modeling. The mutants with notably enhanced activities had small or polar residues in the mutated positions. The most active mutant L107G/L108D/F222H displayed a 70-fold enhanced catalytic efficiency with azathioprine. The determination of its structure by X-ray crystallography showed an expanded H-site, suggesting improved accommodation of the transition state for catalysis.

  • 31.
    Musdal, Yaman
    et al.
    Hacettepe University, Turkey.
    Ertan-Bolelli, Tugba
    Bolelli, Kayhan
    Yilmaz, Serap
    Ceyhan, Deniz
    Hegazy, Usama
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Aksoy, Yasemin
    Inhibition of human glutathione transferase P1-1 by novel benzazole derivatives2012Inngår i: Türk Biyokimya Dergisi, ISSN 0250-4685, E-ISSN 1303-829X, Vol. 37, nr 4, s. 431-436Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Objective: Glutathione transferases (GST) are multifunctional enzymes involved in detoxication, drug resistance, cell signaling and apoptosis. The inhibitory effects of novel benzazole derivatives were tested on human GST P1-1 to find new agents for overcoming drug resistance in cancer cells. Methods: GST P1-1 was heterogously expressed in E. coli strain XL-1 Blue and purified using S-hexylglutathione-Sepharose 6B affinity chromatography. The effect of 33 potential inhibitors on enzymatic activity was assayed spectrophotometrically with 1-chloro-2,4-dinitrobenzene (CDNB) as well as with the alternative substrate phenethyl isothiocyanate (PEITC). Results: Compound-18(N-[2-(4-chloro-benzyl)-benzooxazol-5-yl]-4-nitro-benzenesulfonamide) was the most potent inhibitor found with an IC50 value of approximately 10 mu M with respect to CDNB and a somewhat less strong inhibitor (45 % inhibition at 40 mu M) with PEITC as substrate. Compound-18 showed mixed inhibition with GSH and uncompetitive inhibition with CDNB with the K-i values 6.3 +/- 0.7 mu M and 11.8 +/- 3.4 mu M, respectively. Conclusion: Compound-18 is a potent inhibitor of GST P1-1. It may serve as a lead for further chemical modifications for increased potency. Additional studies will elucidate the effects of the inhibitor on cancer cells.

  • 32.
    Musdal, Yaman
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Govindarajan, Sridhar
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants2017Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 30, nr 8, s. 543-549Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Exploring the vicinity around a locus of a protein in sequence space may identify homologs with enhanced properties, which could become valuable in biotechnical and other applications. A rational approach to this pursuit is the use of 'infologs', i.e. synthetic sequences with specific substitutions capturing maximal sequence information derived from the evolutionary history of the protein family. Ninety-five such infolog genes of poplar glutathione transferase were synthesized and expressed in Escherichia coli, and the catalytic activities of the proteins determined with alternative substrates. Sequence-activity relationships derived from the infologs were used to design a second set of 47 infologs in which 90% of the members exceeded wild-type properties. Two mutants, C2 (V55I/E95D/D108E/A160V) and G5 (F13L/C70A/G122E), were further functionally characterized. The activities of the infologs with the alternative substrates 1-chloro-2,4-dinitrobenzene and phenethyl isothiocyanate, subject to different chemistries, were positively correlated, indicating that the examined mutations were affecting the overall catalytic competence without major shift in substrate discrimination. By contrast, the enhanced protein expressivity observed in many of the mutants were not similarly correlated with the activities. In conclusion, small libraries of well-defined infologs can be used to systematically explore sequence space to optimize proteins in multidimensional functional space.

  • 33.
    Musdal, Yaman
    et al.
    Uppsala University, Sweden; Hacettepe University, Turkey.
    Hegazy, Usama M.
    Aksoy, Yasemin
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-12013Inngår i: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 205, nr 1, s. 53-62Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 34.
    Musdal, Yaman
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Substrate specificities of two tau class glutathione transferases inducible by 2,4,6-trinitrotoluene in poplar2015Inngår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, nr 9, s. 1877-1883Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: The genome of poplar (Populus trichocarpa) encodes 81 glutathione transferases (GSTs) annotated in eight distinct classes. The tau class is considered the most versatile in the biotransformation of xenobiotics and is composed of 58 GSTs. Two of the enzymes, GSTU16 and GSTU45, have particular interest since their expression is induced by exposure of poplar tissues to 2,4,6-trinitrotoluene (TNT) and could potentially be involved in the metabolism of this toxic environmental contaminant.

    Results: DNA encoding these GSTs was synthesized and the proteins were heterologously expressed in Escherichia coli and the purified enzymes were characterized.

    Major conclusions: GSTU16 assayed with a number of conventional GST substrates showed the highest specific activity (60 mu mol min(-1) mg(-1)) with phenethyl isothiocyanate, 150-fold higher than that with CDNB. By contrast, GSTU45 showed CDNB as the most active substrate (3.3 mu mol min(-1) mg(-1)) whereas all of the 16 alternative substrates tested yielded significantly lower activities. Homology modeling suggested that the aromatic residues Phe10 and Tyr107 in the active site of GSTU16 are promoting the high activity with PEITC and other substrates with aromatic side-chains. Nonetheless, TNT was a poor substrate for GSTU16 as well as for GSTU45 with a specific activity of 0.05 nmol min(-1) mg(-1) for both enzymes. General significance: GSTU16 and GSTU45 do not play a major role in the degradation of TNT in poplar.

  • 35. Norrgard, Malena A.
    et al.
    Hellman, Ulf
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase2011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 19, s. 16871-16878Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.

  • 36. Norrgard, Malena A.
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-Site Residue Sustaining Catalytic Promiscuity2011Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 412, nr 1, s. 111-120Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The substrate-binding H-site of human glutathione transferase (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 conserved Ile10 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 substrate selectivity profiles in GSTs by a few mutations.

  • 37. Scian, Michele
    et al.
    Le Trong, Isolde
    Mazari, Aslam M. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Atkins, William M.
    Stenkamp, Ronald E.
    Comparison of epsilon- and delta-class glutathione S-transferases: the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster2015Inngår i: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 71, s. 2089-2098Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta-and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5 angstrom resolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon-and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.

  • 38. Shibata, Aya
    et al.
    Nakano, Yukiko
    Ito, Mika
    Araki, Mika
    Zhang, Jie
    Yoshida, Yasuhiko
    Shuto, Satoshi
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    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 reactions2013Inngår i: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 138, nr 24, s. 7326-7330Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 39. Sun, Song
    et al.
    Zhang, Wei
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Andersson, Dan I.
    Evolution of Broad Spectrum beta-Lactam Resistance in an Engineered Metallo-beta-lactamase2013Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 4, s. 2314-2324Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are beta-lactamases, a group of enzymes inactivating beta-lactam antibiotics. Metallo-beta-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of beta-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several beta-lactam antibiotics was constructed and employed as a parental MBL in an experiment to examine how an enzyme can evolve toward increased activity with a variety of beta-lactam antibiotics. We designed and synthesized a mutant library in which the substrate activity profile was varied by randomizing six active site amino acid residues. The library was expressed in Salmonella typhimurium, clones with increased resistance against seven different beta-lactam antibiotics (penicillin G, ampicillin, cephalothin, cefaclor, cefuroxime, cefoperazone, and cefotaxime) were isolated, and the MBL variants were characterized. For the majority of the mutants, bacterial resistance was significantly increased despite marked reductions in both mRNA and protein levels relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of the mutant enzymes were generalists, conferring increased resistance against most of the examined beta-lactams.

  • 40. Tzafestas, Kyriakos
    et al.
    Razalan, Maria M.
    Gyulev, Ivan
    Mazari, Aslam M. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rylott, Elizabeth L.
    Bruce, Neil C.
    Expression of a Drosophila glutathione transferase in Arabidopsis confers the ability to detoxify the environmental pollutant, and explosive, 2,4,6-trinitrotoluene2017Inngår i: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 214, nr 1, s. 294-303Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale and inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up approach. Here, we have characterized a Drosophila melanogaster glutathione transferase (DmGSTE6) which has activity towards TNT. Recombinantly expressed, purified DmGSTE6 produces predominantly 2-glutathionyl-4, 6-dinitrotoluene, and has a 2.5-fold higher Maximal Velocity (Vmax), and five-fold lower Michaelis Constant (Km) than previously characterized TNT-active Arabidopsis thaliana (Arabidopsis) GSTs. Expression of DmGSTE6 in Arabidopsis conferred enhanced resistance to TNT, and increased the ability to remove TNT from contaminated soil relative to wild-type plants. Arabidopsis lines overexpressing TNT-active GSTs AtGST-U24 and AtGST-U25 were compromised in biomass production when grown in the absence of TNT. This yield drag was not observed in the DmGSTE6-expressing Arabidopsis lines. We hypothesize that increased levels of endogenous TNT-active GSTs catalyse excessive glutathionylation of endogenous substrates, depleting glutathione pools, an activity that DmGST may lack. In conclusion, DmGSTE6 has activity towards TNT, producing a compound with potential for further biodegradation. Selecting or manipulating plants to confer DmGSTE6-like activity could contribute towards development of phytoremediation strategies to clean up TNT from polluted military sites.

  • 41. Wisén, Susanne
    et al.
    Sjögren, Tove
    Olin, Birgit
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Purification, crystallization and preliminary X-ray data of the transcription factor NtcA from the cyanobacterium Anabaena PCC 7120.2004Inngår i: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 60, nr Pt 5, s. 923-5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    NtcA is a transcription factor that acts as a global nitrogen regulator in cyanobacteria. Cyanobacteria are photosynthetic prokaryotic organisms, some genera of which can fix nitrogen under conditions of nitrogen deprivation. NtcA from Anabaena PCC 7120 is a dimeric protein that consists of 223 amino acids with a molecular weight of 25 kDa per subunit. It belongs to the cAMP receptor-protein (CAP) family and is involved in the regulation of several of the genes acting in the nitrogen-fixation process. Here, the crystallization and preliminary X-ray data of NtcA are described. The crystallization was made possible by an improved purification method, which provides a stable NtcA protein at concentrations suitable for crystallization. The protein was crystallized using the hanging-drop method. Data were collected to 2.5 A resolution using synchrotron radiation and the crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 69.23, b = 69.23, c = 162.15 A, alpha = beta = gamma = 90 degrees. The phases necessary to solve the structure of NtcA could not be obtained by molecular replacement based on the CAP structure using various models.

  • 42. Zhang, Jie
    et al.
    Shibata, Aya
    Ito, Mika
    Shuto, Satoshi
    Ito, Yoshihiro
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Abe, Hiroshi
    Morgenstern, Ralf
    Synthesis and Characterization of a Series of Highly Fluorogenic Substrates for Glutathione Transferases, a General Strategy2011Inngår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, nr 35, s. 14109-14119Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferases (GSTs) are used in biotechnology applications as fusion partners for facile purification and are also overexpressed in certain tumors. Consequently, there is a need for sensitive detection of the enzymes. Here we describe a general strategy for the synthesis and characterization of novel fluorogenic substrates for GSTs. The substrates were synthesized by introducing an electrophilic sulfonamide linkage to fluorescent molecules containing an amino group [e.g., 2,4-dinitrobenzenesulfonamide (DNs) derivatives of coumarin, cresyl violet, and rhodamine]. The derivatives were essentially nonfluorescent, and upon GST catalyzed cleavage of the dinitrobenzenesulfonamide, free fluorophore is released (and 1-glutathionyl-2,4-dinitrobenzene + SO(2)). All the coumarin-, cresyl violet- and rhodamine-based fluorogenic probes turned out to be good substrates for most GSTs, especially for GSTA(1-1), in terms of strong fluorescence increases (71-1200-fold), high k(cat)/K(m) values (10(4)-10(7) M(-1) s(-1)) and significant rate enhancements (10(6)-10(9)-fold). The substrates were successfully applied to quantitate very low levels of GST activity in cell extracts and DNs-cresyl violet was also successfully applied to the imaging of microsomal MGST(1) activity in living cells. The cresyl violet stained cells retained their fluorescence after fixation, which is a very useful property. In summary, we describe a general and versatile strategy to generate fluorogenic GST substrates, some of them providing the most sensitive assays so far described for GSTs.

  • 43. Zhang, Wei
    et al.
    Dourado, Daniel F. A. R.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Multidimensional epistasis and fitness landscapes in enzyme evolution2012Inngår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 445, s. 39-46Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The conventional analysis of enzyme evolution is to regard one single salient feature as a measure of fitness, expressed in a milieu exposing the possible selective advantage at a given time and location. Given that a single protein may serve more than one function, fitness should be assessed in several dimensions. In the present study we have explored individual mutational steps leading to a triple-point-mutated human GST (glutathione transferase) A2-2 displaying enhanced activity with azathioprine. A total of eight alternative substrates were used to monitor the diverse evolutionary trajectories. The epistatic effects of the imitations on catalytic activity were variable in sign and magnitude and depended on the substrate used, showing that epistasis is a multidimensional quality. Evidently, the multidimensional fitness landscape can lead to alternative trajectories resulting in enzymes optimized for features other than the selectable markers relevant at the origin of the evolutionary process. In this manner the evolutionary response is robust and can adapt to changing environmental conditions.

  • 44. Zhang, Wei
    et al.
    Dourado, Daniel F. A. R.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden; The Scripps Research Institute, USA.
    Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates2015Inngår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, nr 4, s. 742-749Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective. Methods: In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity. Results: Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis. Conclusions: We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs. General significance: The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.

  • 45. Zhang, Wei
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    An improved dual-tube megaprimer approach for multi-site saturation mutagenesis2013Inngår i: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 29, nr 4, s. 667-672Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.

  • 46. Zhang, Wei
    et al.
    Modén, Olof
    Tars, Kaspars
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Structure-Based Redesign of GST A2-2 for Enhanced Catalytic Efficiency with Azathioprine2012Inngår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, nr 3, s. 414-421Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.

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