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Literature summary for 3.1.8.1 extracted from

  • Jacquet, P.; Hiblot, J.; Daude, D.; Bergonzi, C.; Gotthard, G.; Armstrong, N.; Chabriere, E.; Elias, M.
    Rational engineering of a native hyperthermostable lactonase into a broad spectrum phosphotriesterase (2017), Sci. Rep., 7, 16745 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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

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

Metals/Ions Comment Organism Structure
Co2+ required Saccharolobus solfataricus

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Saccharolobus solfataricus Q97VT7 i.e. Saccharolobus solfataricus
-

Substrates and Products (Substrate)

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

Synonyms Comment Organism
lactonase SsoPox
-
Saccharolobus solfataricus
phosphotriesterase
-
Saccharolobus solfataricus
SsoPox
-
Saccharolobus solfataricus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Saccharolobus solfataricus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.3
-
lactonase activity assay at Saccharolobus solfataricus

General Information

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