Mutated hyperthermophilic phosphotriesterases and their uses
View Patent ↗A subject of the present invention is mutated hyperthermophilic phosphotriesterases (PTEs) possessing a lactonase activity, and their uses as bioscavengers within the context of the decontamination of the surfaces of materials, of the skin or mucous membranes, contaminated with organophosphorus compounds, or within the context of the preparation of medicaments which can be used within the context of the prevention or treatment of an external contamination or of an internal poisoning by ingestion or inhalation by organophosphorus compounds, or within the context of the pollution control of water polluted with organophosphorus compounds.
1. A purified mutated hyperthermophilic phosphotriesterase (PTE) having a lactonase activity derived from a hyperthermophilic PTE corresponding to the consensus sequence of SEQ ID NO: 1, said mutated PTE comprising at least one mutation selected from the group consisting of:
substitution of the tyrosine Y in position 98,
substitution of the tyrosine Y in position 100,
substitution of the arginine R in position 224, and
substitution of the cysteine C in position 259,
of SEQ ID NO: 1 by any other natural or non-natural amino acid,
said mutated hyperthermophilic PTE having a lactonase activity that is greater than that of a non-mutated hyperthermophilic PTE from which it is derived.
2. The mutated hyperthermophilic PTE according to claim 1 , derived from the hyperthermophilic PTE of Sulfolobus solfataricus corresponding to the sequence SEQ ID NO: 3, or from the hyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to the sequence SEQ ID NO: 5, said sequences SEQ ID NO: 3 and SEQ ID NO: 5 belonging to the consensus sequence SEQ ID NO: 1, the amino acid in position 2 in SEQ ID NO: 1 being missing from SEQ ID NO: 3.
3. The mutated hyperthermophilic PTE according to claim 1 , comprising at least the following four mutations:
substitution of the tyrosine Y in position 98,
substitution of the tyrosine Y in position 100,
substitution of the arginine R in position 224, and
substitution of the cysteine C in position 259,
of SEQ ID NO: 1 by any other natural or non-natural amino acid.
4. The mutated hyperthermophilic PTE according to claim 1 , further comprising at least one mutation selected from the group consisting of:
substitution of the valine V in position 28,
substitution of the proline P in position 68,
substitution of the threonine T in position 69,
substitution of the leucine L in position 73,
substitution of the aspartate D in position 142,
substitution of the glycine G in position 226,
substitution of the leucine L in position 227,
substitution of the phenylalanine F in position 230,
substitution of the tryptophan W in position 264, and
substitution of the tryptophan W in position 279,
of SEQ ID NO: 1 by any other natural or non-natural amino acid.
5. The mutated hyperthermophilic PTE according to claim 1 , further comprising the following five mutations:
substitution of the valine V in position 28,
substitution of the leucine L in position 73,
substitution of the aspartate D in position 142,
substitution of the glycine G in position 226, and
substitution of the leucine L in position 227,
of SEQ ID NO: 1 by any other natural or non-natural amino acid.
6. The mutated hyperthermophilic PTE according to claim 1 , further comprising the following five mutations:
substitution of the proline P in position 68,
substitution of the threonine T in position 69,
substitution of the phenylalanine F in position 230,
substitution of the tryptophan W in position 264, and
substitution of the tryptophan W in position 279,
of SEQ ID NO: 1 by any other natural or non-natural amino acid.
7. The mutated hyperthermophilic PTE according to claim 1 , wherein the at least one mutation is selected from the group consisting of:
substitution of the tyrosine Y in position 98 by a tryptophan W,
substitution of the tyrosine Y in position 100 by a phenylalanine F,
substitution of the arginine R in position 224 by a histidine H, and
substitution of the cysteine C in position 259 by a leucine L.
8. The mutated hyperthermophilic PTE according to claim 1 , derived from the hyperthermophilic PTE of Sulfolobus solfataricus corresponding to the sequence SEQ ID NO: 3, and comprising at least one mutation selected from the group consisting of:
substitution of the tyrosine Y in position 97,
substitution of the tyrosine Y in position 99,
substitution of the arginine R in position 223, and
substitution of the cysteine C in position 258,
of SEQ ID NO: 3 by any other natural or non-natural amino acid.
9. The mutated hyperthermophilic PTE according to claim 8 , comprising at least the following four mutations:
substitution of the tyrosine Y in position 97,
substitution of the tyrosine Y in position 99,
substitution of the arginine R in position 223, and
substitution of the cysteine C in position 258,
of SEQ ID NO: 3 by any other natural or non-natural amino acid.
10. The mutated hyperthermophilic PTE according to claim 8 , further comprising at least one mutation selected from the group consisting of:
substitution of the valine V in position 27,
substitution of the proline P in position 67,
substitution of the threonine T in position 68,
substitution of the leucine L in position 72,
substitution of the aspartate D in position 141,
substitution of the glycine G in position 225,
substitution of the leucine L in position 226,
substitution of the phenylalanine F in position 229,
substitution of the tryptophan W in position 263, and
substitution of the tryptophan W in position 278,
of SEQ ID NO: 3 by any other natural or non-natural amino acid.
11. The mutated hyperthermophilic PTE according to claim 8 , comprising the following five mutations:
substitution of the valine V in position 27,
substitution of the leucine L in position 72,
substitution of the aspartate D in position 141,
substitution of the glycine G in position 225, and
substitution of the leucine L in position 226,
of SEQ ID NO: 3 by any other natural or non-natural amino acid.
12. The mutated hyperthermophilic PTE according to claim 8 , comprising the following five mutations:
substitution of the proline P in position 67,
substitution of the threonine T in position 68,
substitution of the phenylalanine F in position 229,
substitution of the tryptophan W in position 263, and
substitution of the tryptophan W in position 278,
of SEQ ID NO: 3 by any other natural or non-natural amino acid.
13. The mutated hyperthermophilic PTE according to claim 8 , comprising at least one mutation selected from the group consisting of:
substitution of the tyrosine Y in position 97 by a tryptophan W,
substitution of the tyrosine Y in position 99 by a phenylalanine F,
substitution of the arginine R in position 223 by a histidine H, and
substitution of the cysteine C in position 258 by a leucine.
14. The mutated hyperthermophilic PTE according to claim 8 , comprising:
SEQ ID NO: 7, corresponding to SEQ ID NO: 3 comprising the following four mutations:
substitution of the tyrosine Y in position 97 by a tryptophan W,
substitution of the tyrosine Y in position 99 by a phenylalanine F,
substitution of the arginine R in position 223 by a histidine H,
substitution of the cysteine C in position 258 by a leucine L,
SEQ ID NO: 9, corresponding to SEQ ID NO: 7 additionally comprising the following five mutations:
substitution of the valine V in position 27 by an alanine A,
substitution of the leucine L in position 72 by an isoleucine I,
substitution of the aspartate D in position 141 by a threonine T,
substitution of the glycine G in position 225 by a proline P,
substitution of the leucine L in position 226 by a histidine H, or
SEQ ID NO: 11, corresponding to SEQ ID NO: 9 additionally comprising the following five mutations:
substitution of the proline P in position 67 by a valine V,
substitution of the threonine T in position 68 by a serine S,
substitution of the phenylalanine F in position 229 by a serine S,
substitution of the tryptophan W in position 263 by an alanine A,
substitution of the tryptophan W in position 278 by an isoleucine I.
15. The mutated hyperthermophilic PTE according to claim 8 , comprising at least one mutation corresponding to a substitution of at least one of the amino acids of the following amino acid pairs, the positions of which in SEQ ID NO: 3 are indicated hereafter, by another natural or non-natural amino acid: 2R/314S, 14K/12E, 26R/75D, 26R/42E, 33R/42E, 33R/45E, 55R/52E, 55R/285E, 74R/121D, 81K/42E, 81K/43D, 84K/80E, 109R/113E, 123K/162E, 147K/148D, 151K/148D, 154R/150E, 154R/187E, 154R/188E, 161K/188E, 183R/150E, 183R/187E, 183R/180E, 210K/245D, 215K/214D, 223R/256D, 223R/202D, 234K/204D, 235R/202D, 241R/245D, 245D/244K, 250K/249D, 277R/286D, 292K/298E, and 310K/307E.
16. The mutated hyperthermophilic PTE according to claim 1 , derived from the hyperthermophilic PTE of Sulfolobus acidocaldarius corresponding to the sequence SEQ ID NO: 5, and comprising at least one mutation selected from the group consisting of:
substitution of the tyrosine Y in position 98,
substitution of the tyrosine Y in position 100,
substitution of the arginine R in position 224, and
substitution of the cysteine C in position 259,
of SEQ ID NO: 5 by any other natural or non-natural amino acid.
17. The mutated hyperthermophilic PTE according to claim 16 , comprising at least the following four mutations:
substitution of the tyrosine Y in position 98,
substitution of the tyrosine Y in position 100,
substitution of the arginine R in position 224, and
substitution of the cysteine C in position 259,
of SEQ ID NO: 5 by any other natural or non-natural amino acid.
18. The mutated hyperthermophilic PTE according to claim 16 , further comprising at least one mutation selected from the group consisting of:
substitution of the valine V in position 28,
substitution of the proline P in position 68,
substitution of the threonine T in position 69,
substitution of the leucine L in position 73,
substitution of the aspartate D in position 142,
substitution of the glycine G in position 226,
substitution of the leucine L in position 227,
substitution of the phenylalanine F in position 230,
substitution of the tryptophan W in position 264, and
substitution of the tryptophan W in position 279,
of SEQ ID NO: 5 by any other natural or non-natural amino acid.
19. The mutated hyperthermophilic PTE according to claim 16 , comprising the following five mutations:
substitution of the valine V in position 28,
substitution of the leucine L in position 73,
substitution of the aspartate D in position 142,
substitution of the glycine G in position 226, and
substitution of the leucine L in position 227,
of SEQ ID NO: 5 by any other natural or non-natural amino acid.
20. The mutated hyperthermophilic PTE according to claim 16 , comprising the following five mutations:
substitution of the proline P in position 68,
substitution of the threonine T in position 69,
substitution of the phenylalanine F in position 230,
substitution of the tryptophan W in position 264, and
substitution of the tryptophan W in position 279,
of SEQ ID NO: 5 by any other natural or non-natural amino acid.
21. The mutated hyperthermophilic PTE according to claim 16 , comprising at least one mutation selected from the group consisting of:
substitution of the tyrosine Y in position 98 by a tryptophan W,
substitution of the tyrosine Y in position 100 by a phenylalanine F,
substitution of the arginine R in position 224 by a histidine H, and
substitution of the cysteine C in position 259 by a leucine L.
22. The mutated hyperthermophilic PTE according to claim 16 , comprising:
SEQ ID NO: 13, corresponding to the sequence SEQ ID NO: 5 comprising the following four mutations:
substitution of the tyrosine Y in position 98 by a tryptophan W,
substitution of the tyrosine Y in position 100 by a phenylalanine F,
substitution of the arginine R in position 224 by a histidine H,
substitution of the cysteine C in position 259 by a leucine L,
SEQ ID NO: 15, corresponding to the sequence SEQ ID NO: 13 additionally comprising the following five mutations:
substitution of the valine V in position 28 by an alanine A,
substitution of the leucine L in position 73 by an isoleucine I,
substitution of the aspartate D in position 142 by a threonine T,
substitution of the glycine G in position 226 by a proline P,
substitution of the leucine L in position 227 by a histidine H, or
SEQ ID NO: 17, corresponding to the sequence SEQ ID NO: 15 additionally comprising the following five mutations:
substitution of the proline P in position 68 by a valine V,
substitution of the threonine T in position 69 by a serine S,
substitution of the phenylalanine F in position 230 by a serine S,
substitution of the tryptophan W in position 264 by an alanine A, and
substitution of the tryptophan W in position 279 by an isoleucine I.
23. The mutated hyperthermophilic PTE according to claim 1 ,
wherein at least one amino acid involved in salt bridges is modified by substitution or deletion, such that an activation temperature of said mutated hyperthermophilic PTE is reduced compared with the activation temperature of the mutated hyperthermophilic PTE in which the at least one amino acid involved in the salt bridges is unmodified.
24. An isolated nucleic acid encoding the mutated hyperthermophilic phosphotriesterase (PTE) of claim 1 .
25. A vector comprising the nucleic acid according to claim 24 .
26. A host cell transformed by the vector according to claim 25 .
27. A composition, comprising the mutated hyperthermophilic PTE according to claim 1 ,
in combination with a pharmaceutically acceptable vehicle.
28. The composition according to claim 27 , in a form which can be administered by injectable route or by topical route.
29. The mutated hyperthermophilic PTE according to claim 7 , further comprising at least one of the following mutations:
substitution of the valine V at position 28 by an alanine A,
substitution of the proline P at position 68 by a valine V,
substitution of the threonine T at position 69 by a serine S,
substitution of the leucine L at position 73 by an isoleucine I,
substitution of the aspartate D at position 142 by a threonine T,
substitution of the glycine G at position 226 by a proline P,
substitution of the leucine L at position 227 by a histidine H,
substitution of the phenylalanine F at position 230 by a serine S,
substitution of the tryptophan W at position 264 by an alanine A, or
substitution of the tryptophan W at position 279 by an isoleucine I.
30. The mutated hyperthermophilic PTE according to claim 5 , comprising the following five mutations:
substitution of the valine V at position 28 by an alanine A,
substitution of the leucine L at position 73 by an isoleucine I,
substitution of the aspartate D at position 142 by a threonine T,
substitution of the glycine G at position 226 by a proline P, and
substitution of the leucine L at position 227 by a histidine H.
31. The mutated hyperthermophilic PTE according to claim 6 , comprising the following five mutations:
substitution of the proline P at position 68 by a valine V,
substitution of the threonine T at position 69 by a serine S,
substitution of the phenylalanine F at position 230 by a serine S,
substitution of the tryptophan W at position 264 by an alanine A, and
substitution of the tryptophan W at position 279 by an isoleucine I.
32. The mutated hyperthermophilic PTE according to claim 13 , further comprising at least one of the following mutations:
substitution of the valine V at position 27 by an alanine A,
substitution of the proline P at position 67 by a valine V,
substitution of the threonine T at position 68 by a serine S,
substitution of the leucine L at position 72 by an isoleucine I,
substitution of the aspartate D at position 141 by a threonine T,
substitution of the glycine G at position 225 by a proline P,
substitution of the leucine L at position 226 by a histidine H,
substitution of the phenylalanine F at position 229 by a serine S,
substitution of the tryptophan W at position 263 by an alanine A, and
substitution of the tryptophan W at position 278 by an isoleucine I.
33. The mutated hyperthermophilic PTE according to claim 8 , comprising the following four mutations:
substitution of the tyrosine Y in position 97 by a tryptophan W,
substitution of the tyrosine Y in position 99 by a phenylalanine F,
substitution of the arginine R in position 223 by a histidine H, and
substitution of the cysteine C in position 258 by a leucine L (SEQ ID NO: 7).
34. The mutated hyperthermophilic PTE according to claim 33 , comprising the following five mutations:
substitution of the valine V at position 27 by an alanine A,
substitution of the leucine L at position 72 by an isoleucine I,
substitution of the aspartate D at position 141 by a threonine T,
substitution of the glycine G at position 225 by a proline P, and
substitution of the leucine L at position 226 by a histidine H (SEQ ID NO: 9).
35. The mutated hyperthermophilic PTE according to claim 34 , comprising the following five mutations:
substitution of the proline P at position 67 by a valine V,
substitution of the threonine T at position 68 by a serine S,
substitution of the phenylalanine F at position 229 by a serine S,
substitution of the tryptophan W at position 263 by an alanine A, and
substitution of the tryptophan W at position 278 by an isoleucine I (SEQ ID NO: 11).