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Literature summary for 3.1.8.1 extracted from

  • Reeves, T.E.; Wales, M.E.; Grimsley, J.K.; Li, P.; Cerasoli, D.M.; Wild, J.R.
    Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities (2008), Protein Eng. Des. Sel., 21, 405-412.
    View publication on PubMed
Search for more literature for 3.1.8.1

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli DH5 alpha cells Brevundimonas diminuta

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapour diffusion method, in 10% PEG6000, 0.1 M HEPES, pH 7.0, 1% diethyl 4-methylbenzylphosphonate, and 5 mM sodium azide Brevundimonas diminuta

Protein Variants

Protein Variants Comment Organism
H254R the mutant enzyme is not completely denatured by urea concentrations up to 8.5 M even after incubation for 24 h, the mutant is more stable than the wild type enzyme and shows increased activity with demeton-S 9times over that of the wild type enzyme Brevundimonas diminuta
H254R/H257F the mutant enzyme shows reduced activity towards paraoxon and increased activity towards demeton-S compared to the wild type enzyme Brevundimonas diminuta
H254R/H257L the variant has higher turnover numbers for the chemical warfare agents O-ethyl S-diisopropyl aminomethyl methylphosphonothioate and O-isobutyl S-N,N-diethylaminoethyl methylphosphonothioate compared to the wild type enzyme, the demeton-S activity is over 14times higher as that of the wild type enzyme Brevundimonas diminuta
H257L the mutant enzyme shows increased activity towards paraoxon and reduced activity toward demeton-S compared to the wild type enzyme Brevundimonas diminuta

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.05
-
 paraoxon mutant enzyme H254R/H257F, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
0.07
-
 paraoxon mutant enzyme H254R/H257L, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
0.09
-
 paraoxon mutant enzyme H254R, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
0.1
-
 paraoxon wild type enzyme, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
0.3
-
 paraoxon mutant enzyme H257L, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
1.9
-
 demeton-S mutant enzyme H257L, in tripart buffer (pH 8.0) Brevundimonas diminuta
4.2
-
 demeton-S mutant enzyme H254R, in tripart buffer (pH 8.0) Brevundimonas diminuta
4.4
-
 demeton-S wild type enzyme, in tripart buffer (pH 8.0) Brevundimonas diminuta
6.2
-
 demeton-S mutant enzyme H254R/H257L, in tripart buffer (pH 8.0) Brevundimonas diminuta
7.6
-
 demeton-S mutant enzyme H254R/H257F, in tripart buffer (pH 8.0) Brevundimonas diminuta

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ the Co2+ form of OPH is the most active of the metal-liganded enzyme forms Brevundimonas diminuta
Zn2+ the Zn2+ form of OPH is one of the most stable dimeric proteins ever identified Brevundimonas diminuta

Organism

Organism UniProt Comment Textmining
Brevundimonas diminuta P0A434 synonym Pseudomonas diminuta
-

Purification (Commentary)

Purification (Comment) Organism
-
 Brevundimonas diminuta

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
demeton-S + H2O
-
 Brevundimonas diminuta 2-ethylsulfanyl-ethanethiol + diethyl phosphate
-
 ?
diethyl 4-methylbenzylphosphonate + H2O
-
 Brevundimonas diminuta ?
-
 ?
O-ethyl S-(2-diisopropylaminoethyl)methylphosphonothioate + H2O also called VX Brevundimonas diminuta ?
-
 ?
O-isobutyl S-(2-N,N-diethylaminoethyl)methylphosphonothioate + H2O also called VR Brevundimonas diminuta ?
-
 ?
paraoxon + H2O
-
 Brevundimonas diminuta diethylphosphate + 4-nitrophenol
-
 ?

Subunits

Subunits Comment Organism
homodimer x-ray crystallography Brevundimonas diminuta

Synonyms

Synonyms Comment Organism
OP hydrolase
-
 Brevundimonas diminuta
OPH
-
 Brevundimonas diminuta
organophosphorus hydrolase
-
 Brevundimonas diminuta

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.3
-
 demeton-S mutant enzyme H257L, in tripart buffer (pH 8.0) Brevundimonas diminuta
3.5
-
 demeton-S wild type enzyme, in tripart buffer (pH 8.0) Brevundimonas diminuta
32
-
 demeton-S mutant enzyme H254R, in tripart buffer (pH 8.0) Brevundimonas diminuta
34
-
 demeton-S mutant enzyme H254R/H257F, in tripart buffer (pH 8.0) Brevundimonas diminuta
50
-
 demeton-S mutant enzyme H254R/H257L, in tripart buffer (pH 8.0) Brevundimonas diminuta
450
-
 paraoxon mutant enzyme H254R/H257F, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
640
-
 paraoxon mutant enzyme H254R/H257L, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
1200
-
 paraoxon mutant enzyme H254R, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
6900
-
 paraoxon wild type enzyme, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta
8000
-
 paraoxon mutant enzyme H257L, in 20 mM CHES buffer (pH 9.0) at 25°C Brevundimonas diminuta