Re Range,50uC,50uC 8.5 sigma 23,338 6 5.79 411 8 omega 29,806 six.01 212 78 24,225 delta eight.35 unclassified 24,457 39 eight 7.80 49 59 epsilon 25,296 5.98 510 8 26,913 theta eight.91 Calculated Molecular Weight Calculated Isoelectric Point 511 8 Substrate Specificity Steady pH Range Optimum pH class CDNB EPNP 4NPB CDNB ECA GPx CDNB CDNB 4HNE 4NPA CDNB 4HNE GPx CDNB DCA Theta-Class Glutathione Transferase in Silkworm respectively . There’s no corresponding region, such as the residue at position 234, in bmGSTT, which may possibly clarify why it exhibits reduced MedChemExpress Madrasin activity than rat, mouse, and human theta-class GSTs. KS-176 web Recently, the electron-sharing network that contributes to the catalytic activity of GST has been described. Based on an amino acid residue at position 64 that is functionally conserved in the GST classes, this network is usually 
divided into variety I and II classes. The form I electron-sharing network is exemplified by delta-, theta-, omega-, and tau-class GSTs, which include an acidic amino acid residue at position 64, whereas the type II network GSTs possess a polar amino acid residue. Glu66 is conserved in the sequence of bmGSTT; thus, this enzyme resembles a member from the type I network. The electron- 7 Theta-Class Glutathione Transferase in Silkworm bmGSTT mutants WT CDNB K ma kcat b H40A V54A E66A S67A R107A 1.5 0.27 0.18 0.52 0.12 0.23 28 3.9 0.22 12 0.88 0.073 four.3 0.35 0.051 0.96 0.16 0.17 kcat /Kmc GSH K ma kcat b 3.6 0.15 0.041 1.8 0.065 0.036 23115181 11 0.52 0.049 12.9 0.28 0.022 ND ND ND 3.9 0.13 0.033 kcat /Kmc 4NPB K ma kcat b 0.13 0.78 6.0 2.three 3.1 1.3 0.50 three.7 7.four ND ND ND ND ND ND 0.52 1.3 two.four kcat /Kmc Values, except those of kcat /Km, are expressed as suggests 1379592 of three independent experiments. a Expressed in units of mM; bexpressed in units of mmol/L/min; and cexpressed in units of/min/mM. ND represents `not detected’. doi:ten.1371/journal.pone.0097740.t003 sharing network in hGSTT2-2 was proposed to include Ser67 as certainly one of residues involved in the network. The equivalent residue in bmGSTT is conserved. Glu66 and Ser67 in bmGSTT could be part of an electron-sharing network as well as the G-site by way of direct interaction with GSH. As a result, mutation from the residues may well lead to a lower in GSH-conjugation activity. Besides five residues in bmGSTT, there might be other amino acid residues which might be necessary for bmGSTT catalytic activity. In theta-class GSTs, the Ser residue inside the N-terminal domain is conserved and considered vital for activation on the bound GSH. The equivalent residue in bmGSTT is Ser11. In other GST classes, mutagenesis of amino acid residues in electronsharing networks benefits in decreased activity. Investiga- tion of putative catalytic residues utilizing site-directed mutagenesis is now underway in our laboratories. Our final results recommend that bmGSTT could possibly play a part in detoxification of xenobiotics in B. mori. Together with bmGSTT, the roles of other GSTs in B. mori needs to be further examined to understand the mechanisms underlying insecticide detoxification. In turn, such research will aid the 
design and implementation of insecticide-resistance management methods for agricultural pests. Author Contributions Conceived and made the experiments: KY NY. Performed the experiments: MDTH NY KY. Analyzed the data: MDTH NY KY. Contributed reagents/materials/analysis tools: MDTH NY KY. Wrote the paper: KY. References 1. Oakley A Glutathione transferases: a structural perspective. Drug Metab Rev 43: 138151. 2. Board PG, Menon D Glutathione t.Re Range,50uC,50uC 8.five sigma 23,338 six five.79 411 eight omega 29,806 six.01 212 78 24,225 delta eight.35 unclassified 24,457 39 8 7.80 49 59 epsilon 25,296 5.98 510 eight 26,913 theta 8.91 Calculated Molecular Weight Calculated Isoelectric Point 511 8 Substrate Specificity Stable pH Variety Optimum pH class CDNB EPNP 4NPB CDNB ECA GPx CDNB CDNB 4HNE 4NPA CDNB 4HNE GPx CDNB DCA Theta-Class Glutathione Transferase in Silkworm respectively . There is absolutely no corresponding area, which includes the residue at position 234, in bmGSTT, which may explain why it exhibits reduce activity than rat, mouse, and human theta-class GSTs. Not too long ago, the electron-sharing network that contributes towards the catalytic activity of GST has been described. Depending on an amino acid residue at position 64 that is functionally conserved within the GST classes, this network is often divided into form I and II classes. The form I electron-sharing network is exemplified by delta-, theta-, omega-, and tau-class GSTs, which include an acidic amino acid residue at position 64, whereas the form II network GSTs have a polar amino acid residue. Glu66 is conserved in the sequence of bmGSTT; as a result, this enzyme resembles a member of the kind I network. The electron- 7 Theta-Class Glutathione Transferase in Silkworm bmGSTT mutants WT CDNB K ma kcat b H40A V54A E66A S67A R107A 1.five 0.27 0.18 0.52 0.12 0.23 28 3.9 0.22 12 0.88 0.073 four.3 0.35 0.051 0.96 0.16 0.17 kcat /Kmc GSH K ma kcat b three.6 0.15 0.041 1.eight 0.065 0.036 23115181 11 0.52 0.049 12.9 0.28 0.022 ND ND ND three.9 0.13 0.033 kcat /Kmc 4NPB K ma kcat b 0.13 0.78 six.0 2.three 3.1 1.three 0.50 3.7 7.4 ND ND ND ND ND ND 0.52 1.3 2.four kcat /Kmc Values, except these of kcat /Km, are expressed as means 1379592 of three independent experiments. a Expressed in units of mM; bexpressed in units of mmol/L/min; and cexpressed in units of/min/mM. ND represents `not detected’. doi:10.1371/journal.pone.0097740.t003 sharing network in hGSTT2-2 was proposed to include Ser67 as certainly one of residues involved in the network. The equivalent residue in bmGSTT is conserved. Glu66 and Ser67 in bmGSTT might be aspect of an electron-sharing network plus the G-site by way of direct interaction with GSH. Hence, mutation from the residues may perhaps lead to a lower in GSH-conjugation activity. Besides 5 residues in bmGSTT, there may be other amino acid residues which might be critical for bmGSTT catalytic activity. In theta-class GSTs, the Ser residue in the N-terminal domain is conserved and viewed as significant for activation of the bound GSH. The equivalent residue in bmGSTT is Ser11. In other GST classes, mutagenesis of amino acid residues in electronsharing networks benefits in decreased activity. Investiga- tion of putative catalytic residues applying site-directed mutagenesis is now underway in our laboratories. Our final results suggest that bmGSTT could play a role in detoxification of xenobiotics in B. mori. Together with bmGSTT, the roles of other GSTs in B. mori need to be additional examined to understand the mechanisms underlying insecticide detoxification. In turn, such research will help the design and implementation of insecticide-resistance management methods for agricultural pests. Author Contributions Conceived and made the experiments: KY NY. Performed the experiments: MDTH NY KY. Analyzed the information: MDTH NY KY. Contributed reagents/materials/analysis tools: MDTH NY KY. Wrote the paper: KY. References 1. Oakley A Glutathione transferases: a structural viewpoint. Drug Metab Rev 43: 138151. two. Board PG, Menon D Glutathione t.
