Re Range,50uC,50uC 8.5 sigma 23,338 six five.79 411 8 omega 29,806 6.01 212 78 24,225 delta 8.35 unclassified 24,457 39 8 7.80 49 59 epsilon 25,296 5.98 510 8 26,913 theta eight.91 Calculated Molecular Weight Calculated Isoelectric Point 511 eight 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, including the residue at position 234, in bmGSTT, which might explain why it exhibits lower activity than rat, mouse, and human theta-class GSTs. Lately, the electron-sharing network that contributes for the catalytic activity of GST has been described. Determined by an amino acid residue at position 64 which is functionally conserved in the GST classes, this network can be divided into sort I and II classes. The type I 
electron-sharing network is exemplified by delta-, theta-, omega-, and tau-class GSTs, which contain an acidic amino acid residue at position 64, whereas the sort II network GSTs have a polar amino acid residue. Glu66 is conserved in the sequence of bmGSTT; therefore, this enzyme resembles a member with the form 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 three.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 3.9 0.13 0.033 kcat /Kmc 4NPB K ma kcat 
b 0.13 0.78 six.0 2.3 3.1 1.three 0.50 3.7 7.four ND ND ND ND ND ND 0.52 1.3 two.four kcat /Kmc Values, except these of kcat /Km, are expressed as signifies 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 inside the network. The equivalent residue in bmGSTT is conserved. Glu66 and Ser67 in bmGSTT might be part of an electron-sharing network along with the G-site through direct interaction with GSH. Therefore, mutation in the residues may perhaps lead to a lower in GSH-conjugation activity. Apart from five residues in bmGSTT, there might be other amino acid residues which are crucial for bmGSTT catalytic activity. In theta-class GSTs, the Ser residue in the N-terminal domain is conserved and regarded as crucial for activation with the bound GSH. The equivalent residue in bmGSTT is Ser11. In other GST classes, mutagenesis of amino acid residues in electronsharing networks results in decreased activity. Investiga- tion of putative catalytic residues utilizing site-directed mutagenesis is now underway in our laboratories. Our outcomes suggest that bmGSTT may well play a role in detoxification of xenobiotics in B. mori. Collectively with bmGSTT, the roles of other GSTs in B. mori ought to be additional examined to know the mechanisms underlying insecticide detoxification. In turn, such studies will aid the style and implementation of insecticide-resistance management techniques for agricultural pests. Author Contributions Conceived and designed 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 point of view. Drug Metab Rev 43: 138151. 2. Board PG, Menon D Glutathione t.Re Range,50uC,50uC 8.5 sigma 23,338 six five.79 411 8 omega 29,806 6.01 212 78 24,225 delta 8.35 unclassified 24,457 39 8 7.80 49 59 epsilon 25,296 five.98 510 eight 26,913 theta 8.91 Calculated Molecular Weight Calculated Isoelectric Point 511 8 Substrate Specificity Stable 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 is absolutely no corresponding region, like the residue at position 234, in bmGSTT, which may possibly clarify 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. Determined by an amino acid residue at position 64 that may be functionally conserved in the GST classes, this network is usually divided into kind 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 kind II network GSTs have a polar amino acid residue. Glu66 is conserved inside the sequence of bmGSTT; therefore, this enzyme resembles a member of the form 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 4.3 0.35 0.051 0.96 0.16 0.17 kcat /Kmc GSH K ma kcat b three.six 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 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 three.7 7.4 ND ND ND ND ND ND 0.52 1.3 2.four kcat /Kmc Values, except those of kcat /Km, are expressed as indicates 1379592 of 3 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 within the network. The equivalent residue in bmGSTT is conserved. Glu66 and Ser67 in bmGSTT may very well be aspect of an electron-sharing network and the G-site through direct interaction with GSH. Hence, mutation with the residues may perhaps lead to a reduce in GSH-conjugation activity. Aside from five residues in bmGSTT, there could possibly be other amino acid residues that happen to be necessary for bmGSTT catalytic activity. In theta-class GSTs, the Ser residue within the N-terminal domain is conserved and considered essential for activation from the bound GSH. The equivalent residue in bmGSTT is Ser11. In other GST classes, mutagenesis of amino acid residues in electronsharing networks outcomes in decreased activity. Investiga- tion of putative catalytic residues making use of site-directed mutagenesis is now underway in our laboratories. Our results recommend that bmGSTT might play a part in detoxification of xenobiotics in B. mori. Collectively with bmGSTT, the roles of other GSTs in B. mori need to be further examined to understand the mechanisms underlying insecticide detoxification. In turn, such studies will aid the design and style and implementation of insecticide-resistance management tactics 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 perspective. Drug Metab Rev 43: 138151. 2. Board PG, Menon D Glutathione t.