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Bengt Mannervik - One of the best experts on this subject based on the ideXlab platform.

  • residue 234 is a master switch of the alternative substrate activity profile of human and rodent theta class Glutathione Transferase T1 1
    Biochimica et Biophysica Acta, 2010
    Co-Authors: Abeer Shokeer, Bengt Mannervik
    Abstract:

    Abstract Background The Theta class Glutathione Transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate Glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site. Methods Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants. Results The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other. Conclusions H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1. General significance The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.

  • minor modifications of the c terminal helix reschedule the favored chemical reactions catalyzed by theta class Glutathione Transferase T1 1
    Journal of Biological Chemistry, 2010
    Co-Authors: Abeer Shokeer, Bengt Mannervik
    Abstract:

    Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione Transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme.

  • single nucleotide polymorphic variants of human Glutathione Transferase T1 1 differ in stability and functional properties
    Archives of Biochemistry and Biophysics, 2009
    Co-Authors: David P Josephy, Meredith Kent, Bengt Mannervik
    Abstract:

    We have previously expressed hexa-histidine-tagged human Glutathione Transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of Glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.

  • structural basis of the suppressed catalytic activity of wild type human Glutathione Transferase T1 1 compared to its w234r mutant
    Journal of Molecular Biology, 2006
    Co-Authors: Kaspars Tars, Abeer Shokeer, Annakarin Larsson, Bengt Mannervik, B Olin, Gerard J Kleywegt
    Abstract:

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

  • residue 234 in Glutathione Transferase T1 1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates
    Biochemical Journal, 2005
    Co-Authors: Abeer Shokeer, Annakarin Larsson, Bengt Mannervik
    Abstract:

    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.

Abeer Shokeer - One of the best experts on this subject based on the ideXlab platform.

  • residue 234 is a master switch of the alternative substrate activity profile of human and rodent theta class Glutathione Transferase T1 1
    Biochimica et Biophysica Acta, 2010
    Co-Authors: Abeer Shokeer, Bengt Mannervik
    Abstract:

    Abstract Background The Theta class Glutathione Transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate Glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site. Methods Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants. Results The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other. Conclusions H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1. General significance The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.

  • minor modifications of the c terminal helix reschedule the favored chemical reactions catalyzed by theta class Glutathione Transferase T1 1
    Journal of Biological Chemistry, 2010
    Co-Authors: Abeer Shokeer, Bengt Mannervik
    Abstract:

    Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione Transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme.

  • structural basis of the suppressed catalytic activity of wild type human Glutathione Transferase T1 1 compared to its w234r mutant
    Journal of Molecular Biology, 2006
    Co-Authors: Kaspars Tars, Abeer Shokeer, Annakarin Larsson, Bengt Mannervik, B Olin, Gerard J Kleywegt
    Abstract:

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

  • residue 234 in Glutathione Transferase T1 1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates
    Biochemical Journal, 2005
    Co-Authors: Abeer Shokeer, Annakarin Larsson, Bengt Mannervik
    Abstract:

    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.

  • residue 234 in Glutathione Transferase T1 1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates
    30th FEBS Congress, 2005
    Co-Authors: Abeer Shokeer, Annakarin Larsson, Bengt Mannervik
    Abstract:

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

Annakarin Larsson - One of the best experts on this subject based on the ideXlab platform.

  • structural basis of the suppressed catalytic activity of wild type human Glutathione Transferase T1 1 compared to its w234r mutant
    Journal of Molecular Biology, 2006
    Co-Authors: Kaspars Tars, Abeer Shokeer, Annakarin Larsson, Bengt Mannervik, B Olin, Gerard J Kleywegt
    Abstract:

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

  • residue 234 in Glutathione Transferase T1 1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates
    Biochemical Journal, 2005
    Co-Authors: Abeer Shokeer, Annakarin Larsson, Bengt Mannervik
    Abstract:

    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.

  • residue 234 in Glutathione Transferase T1 1 plays a pivotal role in the catalytic activity and the selectivity against alternative substrates
    30th FEBS Congress, 2005
    Co-Authors: Abeer Shokeer, Annakarin Larsson, Bengt Mannervik
    Abstract:

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

David P Josephy - One of the best experts on this subject based on the ideXlab platform.

  • single nucleotide polymorphic variants of human Glutathione Transferase T1 1 differ in stability and functional properties
    Archives of Biochemistry and Biophysics, 2009
    Co-Authors: David P Josephy, Meredith Kent, Bengt Mannervik
    Abstract:

    We have previously expressed hexa-histidine-tagged human Glutathione Transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of Glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.

Agneta Rannug - One of the best experts on this subject based on the ideXlab platform.