Literature summary for 3.1.8.1 extracted from

Schofield, D.A.; Dinovo, A.A.
Generation of a mutagenized organophosphorus hydrolase for the biodegradation of the organophosphate pesticides malathion and demeton-S (2010), J. Appl. Microbiol., 109, 548-557.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3)pLysS cells	Flavobacterium sp.

Protein Variants

Protein Variants	Comment	Organism
A80V/F132C/K185R/D208G/H257W/I274N/R319S	the mutant shows increased specific activity with demethon-S methyl and malathion compared to the wild type enzyme	Flavobacterium sp.
A80V/F132D/K185R/D208G/H257W/I274N/R319S	the mutant shows increased specific activity with demethon-S methyl and malathion compared to the wild type enzyme	Flavobacterium sp.
A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S	the mutant shows increased specific activity with VX, demethon-S methyl and malathion compared to the wild type enzyme	Flavobacterium sp.
A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S	the specific activity of the mutant is improved 26, 35 and 42fold against VX, malathion and demeton-S methyl, respectively, compared to the wild type	Flavobacterium sp.
A80V/K185R/D208G/H257W/I274N/R319S	the mutant shows increased specific activity with demethon-S methyl and malathion compared to the wild type enzyme	Flavobacterium sp.
A80V/K185R/D208G/I274N/R319S	the mutant shows increased specific activity with demethon-S methyl and malathion compared to the wild type enzyme	Flavobacterium sp.
G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S	the mutant displays a 77fold improvement in the specific activity against malathion, and exhibits only about 10% of the specific activity against VX compared to the wild type enzyme	Flavobacterium sp.
G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S	the mutant shows increased specific activity with demethon-S methyl and malathion compared to the wild type enzyme	Flavobacterium sp.

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.29	-	malathion	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.41	-	malathion	wild type enzyme, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.67	-	malathion	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.82	-	malathion	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
1.16	-	malathion	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
1.2	-	demeton-S methyl	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
3.64	-	demeton-S methyl	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
4.18	-	demeton-S methyl	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
8.09	-	demeton-S methyl	wild type enzyme, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
23.31	-	demeton-S methyl	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
40000	-	x * 40000, SDS-PAGE	Flavobacterium sp.

Organism

Organism	UniProt	Comment	Textmining
Flavobacterium sp.	-	-	-
Flavobacterium sp. ATCC 27551	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
nickel agarose affinity column chromatography	Flavobacterium sp.

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.000008	-	wild type enzyme, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00015	-	wild type enzyme, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00019	-	mutant enzyme A80V/F132C/K185R/D208G/H257W/I274N/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00022	-	mutant enzyme A80V/K185R/D208G/H257W/I274N/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00045	-	mutant enzyme A80V/F132D/K185R/D208G/H257W/I274N/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00048	-	mutant enzyme A80V/K185R/D208G/I274N/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00075	-	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00151	-	mutant enzyme A80V/K185R/D208G/I274N/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00414	-	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00429	-	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00472	-	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00509	-	mutant enzyme A80V/K185R/D208G/H257W/I274N/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.00617	-	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.007	-	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, using VX as substrate, in 10 mM N-Tris (hydroxymethyl-3-amino propanesulfonic acid), pH 8.0 , at 25°C	Flavobacterium sp.
0.01159	-	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.01404	-	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.01511	-	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, using malathion as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.0194	-	mutant enzyme A80V/F132D/K185R/D208G/H257W/I274N/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.02677	-	mutant enzyme A80V/F132C/K185R/D208G/H257W/I274N/R319S, using demeton-S methyl as substrate, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
0.0803	-	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, using VX as substrate, in 10 mM N-Tris (hydroxymethyl-3-amino propanesulfonic acid), pH 8.0 , at 25°C	Flavobacterium sp.
0.097	-	wild type enzyme, using VX as substrate, in 10 mM N-Tris (hydroxymethyl-3-amino propanesulfonic acid), pH 8.0 , at 25°C	Flavobacterium sp.
2.5	-	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, using VX as substrate, in 10 mM N-Tris (hydroxymethyl-3-amino propanesulfonic acid), pH 8.0 , at 25°C	Flavobacterium sp.

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
acephate + H2O	-	Flavobacterium sp.	?	-	?
acephate + H2O	-	Flavobacterium sp. ATCC 27551	?	-	?
demeton-S methyl + H2O	-	Flavobacterium sp.	2-ethylsulfanyl-ethanethiol + dimethyl phosphate	-	?
malathion + H2O	-	Flavobacterium sp.	O,O-diethyl phosphorothioate + diethyl 2-mercaptosuccinate	-	?
paraoxon + H2O	-	Flavobacterium sp.	4-nitrophenol + diethyl phosphate	-	?
paraoxon + H2O	-	Flavobacterium sp. ATCC 27551	4-nitrophenol + diethyl phosphate	-	?
phosalone + H2O	-	Flavobacterium sp.	?	-	?
sarin + H2O	-	Flavobacterium sp.	?	-	?
sarin + H2O	-	Flavobacterium sp. ATCC 27551	?	-	?
soman + H2O	-	Flavobacterium sp.	?	-	?
soman + H2O	-	Flavobacterium sp. ATCC 27551	?	-	?
tabun + H2O	-	Flavobacterium sp.	?	-	?
tabun + H2O	-	Flavobacterium sp. ATCC 27551	?	-	?
VX + H2O	i.e. O-ethyl S-[2-diisopropylaminoethyl]methylphosphonothiolate	Flavobacterium sp.	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 40000, SDS-PAGE	Flavobacterium sp.

Synonyms

Synonyms	Comment	Organism
OPH	-	Flavobacterium sp.
organophosphorus hydrolase	-	Flavobacterium sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2.55	-	malathion	wild type enzyme, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
13.16	-	demeton-S methyl	wild type enzyme, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
14.62	-	demeton-S methyl	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
23.01	-	malathion	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
40.63	-	malathion	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
44.92	-	malathion	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
45.95	-	demeton-S methyl	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
48.27	-	demeton-S methyl	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
69.44	-	demeton-S methyl	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
73.29	-	malathion	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
1.63	-	demeton-S methyl	wild type enzyme, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
1.97	-	demeton-S methyl	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
3.5	-	demeton-S methyl	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
6.21	-	malathion	wild type enzyme, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
19.08	-	demeton-S methyl	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
19.83	-	malathion	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
40.23	-	demeton-S methyl	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
49.54	-	malathion	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
109.4	-	malathion	mutant enzyme A80V/I106V/F132D/K185R/D208G/H257W/I274N/S308L/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.
154.9	-	malathion	mutant enzyme G60V/A80V/I106V/F132D/K185R/D208G/H257W/I274N/F306V/R319S, in 50 mM HEPES buffer, pH 7.5, at pH, 25°C	Flavobacterium sp.