Isolated phosphotriesterase polypeptides, polynucleotides encoding same and uses thereof in treating or preventing organophosphate exposure associated damage
A genetically modified polypeptide is disclosed which comprises an amino acid sequence of phosphotriesterase (PTE) having at least twice the catalytic efficiency for a V-type nerve agent as a polypeptide which consists of the sequence as set forth in SEQ ID NO: 1, when assayed under identical conditions.
1. A genetically modified phosphotriesterase (PTE) polypeptide:
(a) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 6;
(b) comprising a Leucine residue at position 203, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5), and
(c) having at least twice the catalytic efficiency for a V-type nerve agent as a PTE polypeptide which consists of the sequence as set forth in SEQ ID NO: 1, when assayed at 25° C. under identical conditions,
wherein said modified PTE polypeptide is devoid of the first 29 amino acids of SEQ ID NO: 5.
2. The genetically modified PTE polypeptide of claim 1 , wherein said V-type nerve agent comprises an Sp isomer.
3. The genetically modified PTE polypeptide of claim 1 , wherein said V-type nerve agent is selected from the group consisting of VX, RVX and CVX.
4. The genetically modified PTE polypeptide of claim 1 , wherein said V-type nerve agent is the Sp isomer of VX.
5. A genetically modified phosphotriesterase (PTE) polypeptide:
(a) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 6;
(b) comprising an Asparagine residue at position 173 where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5), and
(c) having catalytic efficiency k cat /K M greater than 3×10 6 M −1 min −1 for the Sp isomer of RVX, retaining at least 50% of its catalytic activity at 50° C. as its catalytic activity at 25° C., when measured in a cell lysate,
wherein said modified PTE polypeptide is devoid of the first 29 amino acids of SEQ ID NO: 5.
6. The genetically modified PTE polypeptide of claim 5 , having a catalytic activity in the presence of 50 μM of the metal chelator 1,10 phenantroline at least 50% of its catalytic activity in the absence of said metal chelator.
7. A genetically modified PTE polypeptide:
(a) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 6;
(b) comprising the mutation Y309W, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5), and
(c) having at least twice the catalytic efficiency for a V-type nerve agent as a PTE polypeptide which consists of the sequence as set forth in SEQ ID NO: 1, when assayed at 25° C. under identical conditions,
wherein said modified PTE polypeptide is devoid of the first 29 amino acids of SEQ ID NO: 5.
8. The genetically modified PTE polypeptide of claim 1 , wherein the amino acid of PTE at position 271 is glycine or arginine, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
9. The genetically modified PTE polypeptide of claim 8 , wherein said amino acid sequence of PTE further comprises the mutation L272W, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
10. The genetically modified PTE polypeptide of claim 7 , wherein said amino acid sequence of PTE further comprises the mutation A270S and/or A203L, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
11. The genetically modified PTE polypeptide of claim 1 , wherein said amino acid sequence of PTE further comprises the mutations F132E, T173N and H254G, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
12. The genetically modified PTE polypeptide of claim 5 , wherein the amino acid at position 203 of said polypeptide is alanine, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
13. The genetically modified PTE polypeptide of claim 5 , wherein the amino acid at position 203 of said polypeptide is phenylalanine where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
14. The genetically modified PTE polypeptide of claim 5 , wherein said amino acid sequence of PTE comprises the mutation T173N, I106A, F132E and H254G, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
15. The genetically modified PTE polypeptide of claim 5 , further comprising the mutation A203F or A203L, where the coordinates correspond to the PTE having the sequence (SEQ ID NO: 5).
16. The genetically modified PTE polypeptide of claim 1 , comprising an amino acid sequence at least 99% homologous to the sequence as set forth in SEQ ID NO: 2, 13, 15, 19 and 23.
17. The genetically modified PTE polypeptide of claim 5 , comprising an amino acid sequence at least 99% homologous to the sequence as set forth in SEQ ID NO: 4, 8, 21 or 23.
18. An isolated polynucleotide comprising a nucleic acid sequence encoding the PTE polypeptide of claim 1 .
19. A pharmaceutical composition comprising as an active ingredient the isolated PTE polypeptide of claim 1 and a pharmaceutically acceptable carrier.
20. A method of treating an organophosphate exposure associated damage in a subject, comprising administering to the subject a therapeutically effective amount of the isolated PTE polypeptide of claim 1 .
21. An article of manufacture for treating or preventing organophosphate exposure associated damage, the article of manufacture comprising the isolated PTE polypeptide of claim 1 immobilized on to a solid support.
22. A method of detoxifying a surface, the method comprising contacting the surface with the isolated PTE polypeptide of claim 1 , thereby detoxifying the surface.
23. A genetically modified PTE polypeptide comprising an amino acid sequence at least 99% homologous to the sequence as set forth in SEQ ID NO: 2, 3, 11, 13, 15, 17, 19, 25, 27, 29, 31, 33 or 35, wherein said modified PTE polypeptide has catalytic efficiency k cat /K M greater than 3×10 6 M −1 min −1 for the Sp isomer of RVX, retaining at least 50% of its catalytic activity at 50° C. as its catalytic activity at 25° C., when measured in a cell lysate,
wherein said modified PTE polypeptide is devoid of the first 29 amino acids of SEQ ID NO: 5.
24. The PTE polypeptide of claim 23 , wherein said amino acid sequence is as set forth in any one of SEQ ID NO: 2, 3, 11, 13, 15, 17, 19, 25, 27, 29, 31, 33 or 35.
25. A pharmaceutical composition comprising as an active ingredient the isolated PTE polypeptide of claim 23 and a pharmaceutically acceptable carrier.
26. A method of treating an organophosphate exposure associated damage in a subject, comprising administering to the subject a therapeutically effective amount of the isolated PTE polypeptide of claim 23 .
27. An article of manufacture for treating or preventing organophosphate exposure associated damage, the article of manufacture comprising the isolated PTE polypeptide of claim 23 immobilized on to a solid support.
28. A method of detoxifying a surface, the method comprising contacting the surface with the isolated PTE polypeptide of claim 23 , thereby detoxifying the surface.
29. A genetically modified PTE polypeptide, comprising an amino acid sequence at least 99% homologous to the sequence as set forth in SEQ ID NO: 4, 8, 21 or 23 and having catalytic efficiency k cat /K M greater than 3×10 6 M −1 min −1 for the Sp isomer of RVX, retaining at least 50% of its catalytic activity at 50° C. as its catalytic activity at 25° C., when measured in a cell lysate.
30. The PTE polypeptide of claim 29 , wherein said amino acid sequence is as set forth in any one of the sequences SEQ ID NO: 4, 8, 21 or 23.