Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged mutant G137D in Escherichia coli strain BL21(DE3) | Lucilia cuprina |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme mutant G137D, hanging drop vapor diffusion method, the reservoir solution contains 100 mM sodium-acetate, pH 4.6, and 20% PEG 2000 monomethyl ester, 17°C, X-ray diffraction structure determination and analysis at 2.01 A resolution, molecular replacement using the previously determined structure of LcalphaE7, PDB ID 4FNG, as template, model building | Lucilia cuprina |
Protein Variants | Comment | Organism |
---|---|---|
G137D | the naturally occurring mutation (Gly137Asp) in alpha-esterase 7 (LcalphaE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. The Gly137Asp mutation does not significantly alter the geometry of the LcalphaE7 catalytic triad or oxyanion hole, organophosphorous substrate binding structure compared to wild-type, overview. The pH-dependence of the rate-determining dephosphorylation step of the reaction by mutant G137D is not altered compared to the wild-type enzyme | Lucilia cuprina |
additional information | replacement of the catalytic serine with the diethylphosphoserine from the structure of the phosphorylated wild-type enzyme (PDB ID 4FNM), molecular dynamics simulations | Lucilia cuprina |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | stopped-flow kinetics, Michaelis-Menten kinetics | Lucilia cuprina | |
0.0086 | - |
diethylumbelliferyl phosphate | pH 7.5, 25°C, recombinant mutant enzyme G137D | Lucilia cuprina |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Lucilia cuprina | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged mutant G137D from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration | Lucilia cuprina |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
diethylumbelliferyl phosphate + H2O | - |
Lucilia cuprina | diethylumbelliferol + phosphate | - |
? | |
O,O-diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate + H2O | i.e. diazinon | Lucilia cuprina | O,O-diethyl phosphorothioate + 4-methyl-6-(propan-2-yl)pyrimidin-2-ol | - |
? |
Synonyms | Comment | Organism |
---|---|---|
organophosphate hydrolase | - |
Lucilia cuprina |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Lucilia cuprina |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.3 | - |
diethylumbelliferyl phosphate | pH 7.5, 25°C, recombinant mutant enzyme G137D | Lucilia cuprina |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Lucilia cuprina |
General Information | Comment | Organism |
---|---|---|
additional information | analysis of the hydrogen bonding of the catalytic triad, mechanism by which Asp137 confers catalytic activity to enzyme LcalphaE7. In LcalphaE7 Gly137Asp:OP, the only other residues that sampled alternative conformations with significant frequency are Met308 and Phe309, which are adjacent to Asp137. These residues are in different conformations in the structure of the Gly137Asp variant relative to the wild-type enzyme | Lucilia cuprina |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
151.16 | - |
diethylumbelliferyl phosphate | pH 7.5, 25°C, recombinant mutant enzyme G137D | Lucilia cuprina |