Literature summary for 3.1.8.1 extracted from

Mabbitt, P.D.; Correy, G.J.; Meirelles, T.; Fraser, N.J.; Coote, M.L.; Jackson, C.J.
Conformational disorganization within the active site of a recently evolved organophosphate hydrolase limits its catalytic efficiency (2016), Biochemistry, 55, 1408-1417 .

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Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged mutant G137D in Escherichia coli strain BL21(DE3)	Lucilia cuprina

Crystallization (Commentary)

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

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 [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

Organism	UniProt	Comment	Textmining
Lucilia cuprina	-	-	-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms	Comment	Organism
organophosphate hydrolase	-	Lucilia cuprina

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Lucilia cuprina

Turnover Number [1/s]

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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Lucilia cuprina

General Information

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 [mM/s]

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

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Release 2023.1 (January 2023)