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Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

Wongsantichon J, Robinson RC, Ketterman AJ - Biosci. Rep. (2015)

Bottom Line: Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance.The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order.This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138673.

No MeSH data available.


Active site pockets of DmGSTE6 in comparison with dmGSTD1Arrangement of helices in dmGSTD1 (a and c) and DmGSTE6 (b and d) contributes to the shape of active site pockets. Stick representation of glutathione is in white and its interacting residues from the N-terminal and the C-terminal domain are in cyan and green, respectively. Tyr106 in panel c is for comparison only.
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Figure 4: Active site pockets of DmGSTE6 in comparison with dmGSTD1Arrangement of helices in dmGSTD1 (a and c) and DmGSTE6 (b and d) contributes to the shape of active site pockets. Stick representation of glutathione is in white and its interacting residues from the N-terminal and the C-terminal domain are in cyan and green, respectively. Tyr106 in panel c is for comparison only.

Mentions: The current structure also shows that the active site pocket for Epsilon class GSTs is more in a ‘closed’ configuration than that of Delta class. Major contributions to this difference arise from the arrangement of helices, especially the positioning of alpha helix 4 (Figures 4a and 4b). This offers an extra GSH interaction with Arg113 from the C-terminal domain, which is a novel feature and perhaps is unique to Epsilon class GSTs (Figures 4c and 4d). To validate the conservation of this arginine and other GSH-binding residues, all Epsilon and Delta class GSTs were retrieved from complete genomes of D. melanogaster and A. gambiae for protein sequence analysis. Based on previous annotation, this includes 14 Epsilon/12 Delta class GSTs from D. melanogaster [13] and 8 Epsilon/15 Delta class GSTs from A. gambiae [29]. Besides the highly conserved catalytic serine (Ser12), we categorized GSH-binding residues into three different groups namely the GSH glycyl moiety, the GSH glutamyl moiety and the C-terminal domain interactor (Figure 5). The impact of protein–ligand interactions toward the glycyl and glutamyl regions of GSH have been extensively studied for the Delta class GSTs. Amino acid conservation in these positions has been reported to play a significant role in enzyme catalysis, substrate specificity and structural integrity of the enzyme [19,27,28,30]. The histidine that interacts with the glycyl moiety of GSH (His53 in DmGSTE6) is highly conserved in both Delta and Epsilon classes of GSTs (Figure 5a). Amino acid replacement of the equivalent residue to His53 in a Delta class GST exhibits a catalytic efficiency approximately 5200-fold lower than observed for the wild-type [28]. For the GSH glutamyl moiety (Figure 5b), this region of interactions also represents functionally conserved residues in both GST classes, and influences protein folding, enzyme kinetics, the rate-limiting step, GSH ionization and the electron-sharing network in catalysis [19,27,30].). It is of interest that the strictly conserved His69 in Epsilon class, which is involved in GSH glutamyl binding, is also one of the ‘wafer’ histidines in the interface motif. This position has a significant role in GSH-binding affinity, substrate specificity and protein folding. As a result, His69, which links the subunits across the dimeric interface and communicates from one active site to the other, may be an important regulating residue for the Epsilon class of GSTs. The final pair of GSH-binding residues in Epsilon class GSTs lies in the C-terminal domain, Phe106 and Arg113. This region is of particular interest since it lies on alpha helix 4, which is positioned relatively closer to the N-terminal domain compared with the Delta class GSTs. Arg113 is conserved only in the Epsilon class (Figure 5c) and it interacts with the glutamyl part of GSH, suggesting that this residue may play a role in modulating GSH/hydrophobic substrate conjugation. Additionally, structural comparison focusing on alpha helix 4 also shows that Phe108 in GSTE6, though conserved in both insect-specific classes of GST (Figure 2), is close enough for hydrophobic interaction with GSH only in the Epsilon class. Distance from Phe108 CE1 atom of DmGSTE6 to GSH CB1 is 3.5 Å whereas the closest distance between dmGSTD1 and GSH is 5.6 Å from Tyr106 CE2 atom to GSH OE1 atom.


Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

Wongsantichon J, Robinson RC, Ketterman AJ - Biosci. Rep. (2015)

Active site pockets of DmGSTE6 in comparison with dmGSTD1Arrangement of helices in dmGSTD1 (a and c) and DmGSTE6 (b and d) contributes to the shape of active site pockets. Stick representation of glutathione is in white and its interacting residues from the N-terminal and the C-terminal domain are in cyan and green, respectively. Tyr106 in panel c is for comparison only.
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Related In: Results  -  Collection

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Figure 4: Active site pockets of DmGSTE6 in comparison with dmGSTD1Arrangement of helices in dmGSTD1 (a and c) and DmGSTE6 (b and d) contributes to the shape of active site pockets. Stick representation of glutathione is in white and its interacting residues from the N-terminal and the C-terminal domain are in cyan and green, respectively. Tyr106 in panel c is for comparison only.
Mentions: The current structure also shows that the active site pocket for Epsilon class GSTs is more in a ‘closed’ configuration than that of Delta class. Major contributions to this difference arise from the arrangement of helices, especially the positioning of alpha helix 4 (Figures 4a and 4b). This offers an extra GSH interaction with Arg113 from the C-terminal domain, which is a novel feature and perhaps is unique to Epsilon class GSTs (Figures 4c and 4d). To validate the conservation of this arginine and other GSH-binding residues, all Epsilon and Delta class GSTs were retrieved from complete genomes of D. melanogaster and A. gambiae for protein sequence analysis. Based on previous annotation, this includes 14 Epsilon/12 Delta class GSTs from D. melanogaster [13] and 8 Epsilon/15 Delta class GSTs from A. gambiae [29]. Besides the highly conserved catalytic serine (Ser12), we categorized GSH-binding residues into three different groups namely the GSH glycyl moiety, the GSH glutamyl moiety and the C-terminal domain interactor (Figure 5). The impact of protein–ligand interactions toward the glycyl and glutamyl regions of GSH have been extensively studied for the Delta class GSTs. Amino acid conservation in these positions has been reported to play a significant role in enzyme catalysis, substrate specificity and structural integrity of the enzyme [19,27,28,30]. The histidine that interacts with the glycyl moiety of GSH (His53 in DmGSTE6) is highly conserved in both Delta and Epsilon classes of GSTs (Figure 5a). Amino acid replacement of the equivalent residue to His53 in a Delta class GST exhibits a catalytic efficiency approximately 5200-fold lower than observed for the wild-type [28]. For the GSH glutamyl moiety (Figure 5b), this region of interactions also represents functionally conserved residues in both GST classes, and influences protein folding, enzyme kinetics, the rate-limiting step, GSH ionization and the electron-sharing network in catalysis [19,27,30].). It is of interest that the strictly conserved His69 in Epsilon class, which is involved in GSH glutamyl binding, is also one of the ‘wafer’ histidines in the interface motif. This position has a significant role in GSH-binding affinity, substrate specificity and protein folding. As a result, His69, which links the subunits across the dimeric interface and communicates from one active site to the other, may be an important regulating residue for the Epsilon class of GSTs. The final pair of GSH-binding residues in Epsilon class GSTs lies in the C-terminal domain, Phe106 and Arg113. This region is of particular interest since it lies on alpha helix 4, which is positioned relatively closer to the N-terminal domain compared with the Delta class GSTs. Arg113 is conserved only in the Epsilon class (Figure 5c) and it interacts with the glutamyl part of GSH, suggesting that this residue may play a role in modulating GSH/hydrophobic substrate conjugation. Additionally, structural comparison focusing on alpha helix 4 also shows that Phe108 in GSTE6, though conserved in both insect-specific classes of GST (Figure 2), is close enough for hydrophobic interaction with GSH only in the Epsilon class. Distance from Phe108 CE1 atom of DmGSTE6 to GSH CB1 is 3.5 Å whereas the closest distance between dmGSTD1 and GSH is 5.6 Å from Tyr106 CE2 atom to GSH OE1 atom.

Bottom Line: Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance.The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order.This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore 138673.

No MeSH data available.