Pon polypeptides, polynucleotides encoding same and compositions and methods utilizing same

Abstract

Isolated polynucleotides and polypeptides encoded therefrom are provided. These include mutated PON enzymes with increased, modified or substantially the same substrate specificity as compared to respective wild-type PON. Also provided are kits and methods using these enzymes.

1 . An isolated polypeptide comprising an amino acid sequence of a PON enzyme, wherein an amino acid sequence of said PON enzyme includes an amino acid substitution corresponding to L69G/V/I, F222S and/or T332S of human PON1. 
     
     
         2 . The isolated polypeptide of  claim 1 , comprising the amino acid sequence as set forth in SEQ ID NO: 50, 52, 53, and 56. 
     
     
         3 . The isolated polypeptide of  claim 1 , comprising the amino acid sequence encoded by the nucleic acid sequence selected from the group consisting of SEQ ID NO: 45, 81, 82, 83, 84, 85, 86 and 87. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein said amino acid sequence of said PON enzyme includes at least two amino acid substitutions. 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein said amino acid sequence of said PON enzyme includes substitutions at amino acid coordinates corresponding to L69G/V/I, F222S and T332S of said human PON1. 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein said amino acid sequence of said PON enzyme includes substitutions at amino acid coordinates corresponding to F222S and T332S of said human PON1. 
     
     
         9 . A nucleic acid construct comprising an isolated polynucleotide encoding the isolated polypeptide of  claim 1 . 
     
     
         10 . A host cell comprising the nucleic acid construct of  claim 9 . 
     
     
         11 . A pharmaceutical composition comprising a therapeutically effective amount of the isolated polypeptide of  claim 1 . 
     
     
         12 . A method of treating or preventing a PON-related disease or condition, the method is effected by administering to a subject in need a therapeutically effective amount of an isolated polypeptide comprising an amino acid sequence of a mutated PON1 enzyme, wherein an amino acid sequence of said mutated PON enzyme includes at an amino acid substitution in at least one amino acid selected from the group consisting of amino acids 69, 74, 75, 76, 78, 115, 134, 190, 192, 193, 196, 222, 240, 291, 292, 293, 332, and 346 of a human PON1, said mutated PON1 enzyme being characterized by an increased substrate specificity to at least one known PON substrate as compared to a respective wild-type PON. 
     
     
         13 . The method of  claim 12 , wherein the disease or condition is selected from the group consisting of hyperlipidemia, atherosclerosis, neurological disease, cancer and organophosphate poisoning. 
     
     
         14 . The method of  claim 12 , wherein said neurological disease is selected from the group consisting of Alzheimer's disease, neurofibromatosis, Huntington's disease, depression, amyotrophic lateral sclerosis, multiple sclerosis, stroke, Parkinson's disease, or multi-infarct dementia. 
     
     
         15 . The method of  claim 12 , wherein said isolated polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 49, 50, 52, 53, 54, 56, 57, 58, 59, 60, 61, 63, 65, 67, 69, 71, 73, 75 or 77. 
     
     
         16 . The method of  claim 12 , wherein said substrate specificity is at least 3 fold higher k at /K M  ratio for organophosphate hydrolysis with respect to said wild type PON. 
     
     
         17 . The method of  claim 12 , wherein said isolated polypeptide exhibits an increased thiolactonase activity and a decreased phosphotriesterase activity. 
     
     
         18 . The method of  claim 12 , wherein said amino acid sequence of said mutated PON enzyme includes at least two amino acid substitutions. 
     
     
         19 . The method of  claim 12 , wherein said amino acid sequence of said mutated PON enzyme includes substitutions at amino acid coordinates 69, 115, 134, 222 and 332 of said human PON. 
     
     
         20 . The method of  claim 12 , wherein said human PON is human PON1.