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Literature summary 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)

EC Number Cloned (Comment) Organism
3.1.1.81 expressed in Escherichia coli BL21(DE3) cells Saccharolobus solfataricus
3.1.8.1 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)

EC Number Crystallization (Comment) Organism
3.1.1.81 hanging drop vapor diffusion method, using 20-30% (w/v) PEG 8000 and 50 mM Tris-HCl buffer (pH 8.0) Saccharolobus solfataricus
3.1.8.1 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

EC Number Protein Variants Comment Organism
3.1.1.81 C258L/I261F/W263A the mutant exhibits enhanced phosphotriesterase activity compared to the wild type enzyme Saccharolobus solfataricus
3.1.1.81 F46L/C258A/W263M/I280T the mutant exhibits enhanced phosphotriesterase activity compared to the wild type enzyme Saccharolobus solfataricus
3.1.1.81 L72I/Y99F/I122L/L228M/F229S/W263L the mutant exhibits enhanced phosphotriesterase activity compared to the wild type enzyme Saccharolobus solfataricus
3.1.1.81 V27A/I76T/Y97W/Y99F/L130P/L226V the mutant exhibits enhanced phosphotriesterase activity compared to the wild type enzyme Saccharolobus solfataricus
3.1.1.81 V27A/Y97W/L228M/W263M the mutant demonstrates a large increase in catalytic efficiencies over the wild-type enzyme, with increases of 2210fold, 163fold, 58fold, 16fold against methyl parathion, malathion, ethyl paraoxon, and methyl paraoxon, respectively Saccharolobus solfataricus
3.1.8.1 C258A site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 C258L site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 C258L/I261F/W263A site-directed mutagenesis, mutant alphasA1, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 F46L/C258A/W263M/I280T site-directed mutagenesis, mutant alphasA6, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 I767T site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 L130P site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 L228M site-directed mutagenesis Saccharolobus solfataricus
3.1.8.1 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
3.1.8.1 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
3.1.8.1 V27A site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 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
3.1.8.1 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
3.1.8.1 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
3.1.8.1 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
3.1.8.1 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
3.1.8.1 W263I site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 W263L site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 W263N site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus
3.1.8.1 Y97W site-directed mutagenesis Saccharolobus solfataricus
3.1.8.1 Y99F site-directed mutagenesis, active site mutant, the mutant shows altered substrate specificity compared to the wild-type enzyme Saccharolobus solfataricus

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.1.1.81 fensulfothion
-
 Saccharolobus solfataricus
3.1.8.1 fensulfothion inhibits the wild-type enzyme, while some enzyme mutants are also capable of degrading fensulfothion as substrate Saccharolobus solfataricus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.1.8.1 Co2+ required Saccharolobus solfataricus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.1.1.81 an N-acyl-L-homoserine lactone + H2O Saccharolobus solfataricus
-
 an N-acyl-L-homoserine
-
 ?
3.1.8.1 diethyl-paraoxon + H2O Saccharolobus solfataricus
-
 diethyl phosphate + 4-nitrophenol
-
 ?
3.1.8.1 diethyl-parathion + H2O Saccharolobus solfataricus
-
 diethyl thiophosphate + 4-nitrophenol
-
 ?
3.1.8.1 dimethyl-paraoxon + H2O Saccharolobus solfataricus
-
 dimethyl phosphate + 4-nitrophenol
-
 ?
3.1.8.1 dimethyl-parathion + H2O Saccharolobus solfataricus
-
 dimethyl thiophosphate + 4-nitrophenol
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.1.1.81 Saccharolobus solfataricus Q97VT7 Sulfolobus solfataricus
-
3.1.8.1 Saccharolobus solfataricus Q97VT7 i.e. Saccharolobus solfataricus
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.1.81 ammonium sulfate precipitation and Superdex 75 gel filtration Saccharolobus solfataricus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.1.81 an N-acyl-L-homoserine lactone + H2O
-
 Saccharolobus solfataricus an N-acyl-L-homoserine
-
 ?
3.1.1.81 additional information no activity with coumaphos, fensulfothion, fenitrothion, diazinon, chlorpyrifos, and ethyl parathion. Low activity with malathion, methyl parathion methyl paraoxon, and ethyl paraoxon Saccharolobus solfataricus ?
-
 ?
3.1.1.81 N-3-oxododecanoyl-L-homoserine lactone + H2O
-
 Saccharolobus solfataricus N-3-oxododecanoyl-L-homoserine
-
 ?
3.1.1.81 undecanoic-delta-lactone + H2O
-
 Saccharolobus solfataricus 5-hydroxyundecanoic acid
-
 ?
3.1.1.81 undecanoic-gamma-lactone + H2O
-
 Saccharolobus solfataricus 4-hydroxyundecanoic acid
-
 ?
3.1.8.1 chlorpyrifos + H2O low activity, cf. EC 3.1.8.2 Saccharolobus solfataricus O,O-diethylphosphorothioate + 3,5,6-trichloropyridin-2-ol
-
 ?
3.1.8.1 coumaphos + H2O cf. EC 3.1.8.2 Saccharolobus solfataricus O,O-diethylphosphorothioate + 3-chloro-7-hydroxy-4-methyl-2H-chromen-2-one
-
 ?
3.1.8.1 diethyl-paraoxon + H2O
-
 Saccharolobus solfataricus diethyl phosphate + 4-nitrophenol
-
 ?
3.1.8.1 diethyl-parathion + H2O
-
 Saccharolobus solfataricus diethyl thiophosphate + 4-nitrophenol
-
 ?
3.1.8.1 dimethyl-paraoxon + H2O
-
 Saccharolobus solfataricus dimethyl phosphate + 4-nitrophenol
-
 ?
3.1.8.1 dimethyl-parathion + H2O
-
 Saccharolobus solfataricus dimethyl thiophosphate + 4-nitrophenol
-
 ?
3.1.8.1 fenitrothion + H2O
-
 Saccharolobus solfataricus O,O-diethylphosphorothioate + 3-methyl-4-nitrophenol
-
 ?
3.1.8.1 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
-
 ?
3.1.8.1 malathion + H2O low activity Saccharolobus solfataricus O,O-diethylphosphorothioate + diethyl 2-mercaptosuccinate
-
 ?
3.1.8.1 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

EC Number Synonyms Comment Organism
3.1.1.81 POX
-
 Saccharolobus solfataricus
3.1.8.1 lactonase SsoPox
-
 Saccharolobus solfataricus
3.1.8.1 phosphotriesterase
-
 Saccharolobus solfataricus
3.1.8.1 SsoPox
-
 Saccharolobus solfataricus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.8.1 25
-
 assay at Saccharolobus solfataricus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.8.1 8.3
-
 lactonase activity assay at Saccharolobus solfataricus

General Information

EC Number General Information Comment Organism
3.1.8.1 additional information active site residues are Y99, L228, F229 and W263. Structure-activity analysis of wild-type and mutant enzymes, overview Saccharolobus solfataricus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.1.1.81 0.141
-
 N-3-oxododecanoyl-L-homoserine lactone mutant enzyme C258L/I261F/W263A, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 0.584
-
 N-3-oxododecanoyl-L-homoserine lactone mutant enzyme L72I/Y99F/I122L/L228M/F229S/W263L, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 0.905
-
 undecanoic-gamma-lactone mutant enzyme V27A/I76T/Y97W/Y99F/L130P/L226V, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 1.63
-
 undecanoic-gamma-lactone mutant enzyme C258L/I261F/W263A, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 1.78
-
 N-3-oxododecanoyl-L-homoserine lactone mutant enzyme F46L/C258A/W263M/I280T, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 2.36
-
 undecanoic-gamma-lactone wild type enzyme, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 2.55
-
 undecanoic-delta-lactone wild type enzyme, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 2.55
-
 undecanoic-delta-lactone mutant enzyme C258L/I261F/W263A, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 5.33
-
 undecanoic-delta-lactone mutant enzyme V27A/I76T/Y97W/Y99F/L130P/L226V, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 7.5
-
 undecanoic-delta-lactone mutant enzyme V27A/Y97W/L228M/W263M, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 16
-
 undecanoic-gamma-lactone mutant enzyme V27A/Y97W/L228M/W263M, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 31.6
-
 N-3-oxododecanoyl-L-homoserine lactone wild type enzyme, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 360
-
 undecanoic-delta-lactone mutant enzyme L72I/Y99F/I122L/L228M/F229S/W263L, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 440
-
 undecanoic-gamma-lactone mutant enzyme F46L/C258A/W263M/I280T, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 573
-
 undecanoic-gamma-lactone mutant enzyme L72I/Y99F/I122L/L228M/F229S/W263L, at pH 8.0 and 25°C Saccharolobus solfataricus
3.1.1.81 9650
-
 undecanoic-delta-lactone mutant enzyme F46L/C258A/W263M/I280T, at pH 8.0 and 25°C Saccharolobus solfataricus