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Literature summary for 3.1.8.1 extracted from
- An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination (2016), Biotechnol. Bioeng., 113, 724-734 .
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| commercial soap LM1 | wild-type SsoPox, at 70°C, 13fold enhancement of hydrolytic activity in the presence of 0.05% LM1, and for mutant C258L/I261F/W263A at 65°C 1.3fold enhancement | Saccharolobus solfataricus | |
| SDS | wild-type SsoPox, at 70°C, 17fold enhancement of hydrolytic activity in the presence of 0.025% SDS, and for mutant C258L/I261F/W263A at 65°C 2fold enhancement | Saccharolobus solfataricus |  |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene ssopox and gene ssopox-pte, sequence comparisons, recombinant expression of wild-type, point mutation, and chimeric mutant enzymes in Escherichia coli strains TOP10 and BL21(DE3) | Saccharolobus solfataricus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| C258L/I261F/W263A | site-directed mutagenesis, in vitro evolution, the mutant activity is promiscuous. The combination of C258L, I261F, and W263A mutations in the SsoPox triple mutant improves the hydrolytic specific activity in terms of kcat/KM toward paraoxon 12fold, the kcat 294fold compared to wild-type SsoPox, while the KM value increases | Saccharolobus solfataricus |
| D141T | site-directed mutagenesis, the mutant enzyme shows increased phosphotriesterase activity compared to the wild-type | Saccharolobus solfataricus |
| additional information | evolution of a lactonase into a phosphotriesterase, semi-rational engineering approach is used to design an efficient and thermostable organophosphate hydrolase, starting from enzyme SsoPox from Sulfolobus solfataricus as a lactonase scaffold. In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A is obtained which, retaining its inherent stability, shows an enhancement of its hydrolytic activity on paraoxon up to 300fold. The mutant is tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. Construction of a chimeric gene ssopox-pte by insertion of 16 conserved residues of pte gene in the ssopox sequence. Recombination by DNA StEP between ssopox-pte chimera and ssopox gene | Saccharolobus solfataricus |
| V27A | site-directed mutagenesis, the mutant enzyme shows increased phosphotriesterase activity compared to the wild-type | Saccharolobus solfataricus |
| V27A/D141T | site-directed mutagenesis, the mutant enzyme shows increased phosphotriesterase activity compared to the wild-type | Saccharolobus solfataricus |
| V27A/Y97W/Y99F | site-directed mutagenesis, the mutant enzyme shows increased phosphotriesterase activity compared to the wild-type | Saccharolobus solfataricus |
| W263F | site-directed mutagenesis, the W263 residue is previously demonstrated to be involved in the formation of an hydrophobic channel for the substrate leaving group, the mutant enzyme shows increased phosphotriesterase activity compared to the wild-type | Saccharolobus solfataricus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetics, kinetic analysis of phosphotriesterase activity | Saccharolobus solfataricus | |
| 0.107 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 25°C, with 0.025% SDS | Saccharolobus solfataricus | |
| 0.325 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C, with 0.025% SDS | Saccharolobus solfataricus | |
| 0.38 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 25°C | Saccharolobus solfataricus | |
| 1.586 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C | Saccharolobus solfataricus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Co2+ | activates, required | Saccharolobus solfataricus | |
| additional information | the enzyme has a binuclear metal-centre. The activity depends on the presence of divalent metal cations, the highest activity is observed with Co2+. The binuclear centre is used to activate a bridging water molecule to a hydroxide ion and the substrate for nucleophilic attack by polarizing the phosphoryl-oxygen bond. The nucleophilic bridging hydroxide ion attacks the electrophilic centre (phosphorus or carbon) via a SN2 mechanism, forming transition states that bridge the two metals | Saccharolobus solfataricus |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | Saccharolobus solfataricus | the phosphotriesterase-like lactonase enzyme is bifunctional showing lactonase (EC 3.1.1.81) and phosphotriesterase (EC 3.1.8.1 and 3.1.8.2) activities | ? | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Saccharolobus solfataricus | Q97VT7 | i.e. Saccharolobus solfataricus | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.42 | - |
purified recombinant enzyme mutant W263F, pH 8.5, 65°C, substrate diethyl-paraoxon | Saccharolobus solfataricus |
| 12.8 | - |
purified recombinant enzyme mutant W263F, pH 8.5, 65°C, substrate diethyl-paraoxon | Saccharolobus solfataricus |
| 123.6 | - |
purified recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C, substrate diethyl-paraoxon | Saccharolobus solfataricus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| cyclohexylmethylphosphonofluoridate + H2O | i.e. cyclosarin | Saccharolobus solfataricus | ? | - |
? | |
| diethyl-paraoxon + H2O | - |
Saccharolobus solfataricus | diethyl phosphate + 4-nitrophenol | - |
? | |
| diethyl-parathion + H2O | - |
Saccharolobus solfataricus | diethyl thiophosphate + 4-nitrophenol | - |
? | |
| dimethyl-paraoxon + H2O | - |
Saccharolobus solfataricus | dimethyl phosphate + 4-nitrophenol | - |
? | |
| dimethyl-parathion + H2O | - |
Saccharolobus solfataricus | dimethyl thiophosphate + 4-nitrophenol | - |
? | |
| ethyl dimethylphosphoramidocyanidate + H2O | i.e. tabun | Saccharolobus solfataricus | ? | - |
? | |
| additional information | the phosphotriesterase-like lactonase enzyme is bifunctional showing lactonase (EC 3.1.1.81) and phosphotriesterase (EC 3.1.8.1 and 3.1.8.2) activities | Saccharolobus solfataricus | ? | - |
? | |
| additional information | substrate docking analysis. The C258 residue in the active site is involved in an interaction with the oxygen atom from the amide in the lactone analogue | Saccharolobus solfataricus | ? | - |
? | |
| O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate + H2O | i.e. VX | Saccharolobus solfataricus | ? | - |
? | |
| O-isopropyl methylphosphonofluoridate + H2O | i.e. sarin | Saccharolobus solfataricus | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| lactonase/phosphotriesterase | - |
Saccharolobus solfataricus |
| organophosphate hydrolase | - |
Saccharolobus solfataricus |
| phosphotriesterase-like lactonase | - |
Saccharolobus solfataricus |
| PHP | - |
Saccharolobus solfataricus |
| PLL | - |
Saccharolobus solfataricus |
| SsoPox | - |
Saccharolobus solfataricus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 65 | - |
enzyme mutant C258L/I261F/W263A | Saccharolobus solfataricus |
| 85 | - |
enzyme mutant W263F | Saccharolobus solfataricus |
| 90 | - |
above, wild-type enzyme | Saccharolobus solfataricus |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 65 | - |
purified recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 6.6 h, completely stable | Saccharolobus solfataricus |
| 75 | - |
purified recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, loss of 10% activity after 6.6 h | Saccharolobus solfataricus |
| 85 | - |
purified recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, loss of 30% activity after 6.6 h, t1/2 is 5.8 h | Saccharolobus solfataricus |
| 90 | - |
purified recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, loss of 40% activity after 5.0 h, t1/2 is 2.2 h | Saccharolobus solfataricus |
| 95 | - |
purified recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, loss of 40% activity after 1.5 h, t1/2 is 25 min | Saccharolobus solfataricus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 6.91 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 25°C | Saccharolobus solfataricus | |
| 8.6 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 25°C, with 0.025% SDS | Saccharolobus solfataricus | |
| 31.7 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C, with 0.025% SDS | Saccharolobus solfataricus | |
| 71.05 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C | Saccharolobus solfataricus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | 8.5 | wild-type enzyme and mutant W263F | Saccharolobus solfataricus |
| 8.5 | - |
enzyme mutant C258L/I261F/W263A | Saccharolobus solfataricus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | enzyme SsoPox belongs to the phosphotriesterase-like lactonase (PLL) family of enzymes. SsoPox shares only about 30% sequence identity with phosphotriesterases (PTEs) but all amino acids coordinating the binuclear metal-centre are conserved. The coexistence of lactonase and phosphotriesterase activities has been already reported for many members of PLL family | Saccharolobus solfataricus |
| physiological function | SsoPox is a thermostable phosphotriesterase-like lactonase (PLL) that hydrolyses lactones (primary activity) and, at a lower rate, neurotoxic organophosphorus compounds (promiscuous activity) | Saccharolobus solfataricus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 18.19 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 25°C | Saccharolobus solfataricus | |
| 44.8 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C | Saccharolobus solfataricus | |
| 80.4 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 25°C, with 0.025% SDS | Saccharolobus solfataricus | |
| 97.5 | - |
diethyl-paraoxon | recombinant enzyme mutant C258L/I261F/W263A, pH 8.5, 65°C, with 0.025% SDS | Saccharolobus solfataricus | |
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