Cloned (Comment) | Organism |
---|---|
plasmid library screening, recombinant wild-type and mutant enzymes from Escherichia coli BL21(DE3)-pGro7/GroEL (TaKaRa) chaperone expressing strain by ammonium sulfate fractionation, desalting gel filtration, and gel filtration | Saccharolobus solfataricus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type enzyme and mutant asA6 in open conformation, asA6 in closed conformation, asB5, asC6, asD6, and asA1, hanging drop vapor diffusion method, mixing of 500 nl protein solution, containing protein in 50 mM HEPES, pH 8.0, 150 mM NaCl, and 0.2 mM CoCl2, and 500 nl reservoir solution, containing 20-30% w/v PEG 8000, and 50 mM Tris-HCl, pH 8.0, a few days, 4°C, X-ray diffraction structure determination and analysis at 1.4-2.95 A resolution | Saccharolobus solfataricus |
Protein Variants | Comment | Organism |
---|---|---|
C258A | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
C258L | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
C258L/I261F/W263A | site-directed mutagenesis, mutant alphasA1, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
F46L/C258A/W263M/I280T | site-directed mutagenesis, mutant alphasA6, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
I767T | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
L130P | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
L228M | site-directed mutagenesis | Saccharolobus solfataricus |
L72I/Y99F/I122L/L228M/F229S/W263L | site-directed mutagenesis, mutant alphasC6, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
additional information | the lactonase SsoPox is engineered for higher phosphotriesterase activity using structure-based combinatorial libraries. By comparing structures of enzymes with similar topology, it is possible to redesign, using modelling tools, the active site cavity of SsoPox to mimic as closely as possible that of enzyme BdPTE from Brevundimonas diminuta. Some enzyme mutants are also capable of degrading fensulfothion, which is reported to be an inhibitor for the wild-type enzyme, as well as others that are not substrates of the starting template or previously reported W263 mutants. Structure-based identification of mutations, structure-activity analysis of wild-type and mutant enzymes, overview. Construction of SsoPox monovariants alphasA1, alphasA6, alphasB5, alphasC6 and alphasD6 | Saccharolobus solfataricus |
V27A | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
V27A/I76T/Y97W/Y99F/L130P/L226V | site-directed mutagenesis, mutant alphasB5, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
V27A/I76T/Y97W/Y99F/L130P/L226V/W263I | site-directed mutagenesis, mutant alphasB5 W263I, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
V27A/I76T/Y97W/Y99F/L130P/L226V/W263L | site-directed mutagenesis, mutant alphasB5 W263L, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
V27A/I76T/Y97W/Y99F/L130P/L226V/W263M | site-directed mutagenesis, mutant alphasB5 W263M, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
V27A/Y97W/L228M/W263M | site-directed mutagenesis, the mutant alphasD6 enzyme demonstrates a large increase in catalytic efficiencies compared to the wild-type enzyme, with increases of 2210fold, 163fold, 58fold, and 16fold against methyl-parathion, malathion, ethyl-paraoxon, and methyl-paraoxon, respectively | Saccharolobus solfataricus |
W263I | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
W263L | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
W263N | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
Y97W | site-directed mutagenesis | Saccharolobus solfataricus |
Y99F | site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme | Saccharolobus solfataricus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
fensulfothion | inhibits the wild-type enzyme, while some enzyme mutants are also capable of degrading fensulfothion as substrate | Saccharolobus solfataricus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Co2+ | required | Saccharolobus solfataricus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharolobus solfataricus | Q97VT7 | i.e. Saccharolobus solfataricus | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
chlorpyrifos + H2O | low activity, cf. EC 3.1.8.2 | Saccharolobus solfataricus | O,O-diethylphosphorothioate + 3,5,6-trichloropyridin-2-ol | - |
? | |
coumaphos + H2O | cf. EC 3.1.8.2 | Saccharolobus solfataricus | O,O-diethylphosphorothioate + 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one | - |
? | |
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 | - |
? | |
fenitrothion + H2O | - |
Saccharolobus solfataricus | O,O-diethylphosphorothioate + 3-methyl-4-nitrophenol | - |
? | |
fensulfothion + H2O | some enzyme mutants are also capable of degrading fensulfothion, which is reported to be an inhibitor for the wild-type enzyme, as well as others that are not substrates of the starting template or previously reported W263 mutants | Saccharolobus solfataricus | O,O-diethylphosphorothioate + 4-(methylsulfinyl)phenol | - |
? | |
malathion + H2O | low activity | Saccharolobus solfataricus | O,O-diethylphosphorothioate + diethyl 2-mercaptosuccinate | - |
? | |
additional information | the enzyme shows lactonase activity with N-(3-oxodecanoyl)-L-homoserine lactone, undecanoic-gamma-lactone, and undecanoic-delta-lactone, cf. EC 3.1.1.81 | Saccharolobus solfataricus | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
lactonase SsoPox | - |
Saccharolobus solfataricus |
phosphotriesterase | - |
Saccharolobus solfataricus |
SsoPox | - |
Saccharolobus solfataricus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Saccharolobus solfataricus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.3 | - |
lactonase activity assay at | Saccharolobus solfataricus |
General Information | Comment | Organism |
---|---|---|
additional information | active site residues are Y99, L228, F229 and W263. Structure-activity analysis of wild-type and mutant enzymes, overview | Saccharolobus solfataricus |