Deregulation of glutamine PRPP amidotransferase activity

Abstract

The present invention is directed methods of purine synthesis using a modified amidotransferase, wherein the modified glutamine PRPP amidotransferase has reduced sensitivity to end-product inhibition of synthesizing purine nucleotides.

1 . A method of enhancing purine nucleotide biosynthesis, said method comprising the steps of culturing a host cell that comprises a gene sequence encoding a modified glutamine PRPP amidotransferase, wherein the modified glutamine PRPP amidotransferase has reduced sensitivity to end-product inhibition of synthesizing purine nucleotides.  
     
     
         2 . The method of  claim 1  wherein the modified glutamine PRPP amidotransferase is a modified Bacillus glutamine PRPP amidotransferase, wherein the amino acid sequence of said modified Bacillus amidotransferase differs from that set forth in SEQ. ID NO:1 in that one or more amino acid residues at positions selected from the group consisting of 282, 283, 307, and 347 of SEQ ID NO:1 are substituted with an amino acid not native to said position or positions, and wherein said modified Bacillus amidotransferase is less sensitive to end-product inhibition than is a Bacillus amidotransferase as set forth in SEQ. ID NO:1.  
     
     
         3 . The method of  claim 2  wherein the amino acid substitution reduces synergistic binding of adenine and guanine nucleotides to the amidotransferase.  
     
     
         4 . The method of  claim 1  wherein the gene sequence encoding the glutamine PRPP amidotransferase is operably linked to a constitutive promoter.  
     
     
         5 . The method of  claim 1  wherein the amino acid sequence of said modified amidotransferase differs a corresponding native glutamine PRPP amidotransferase in that one or more amino acid residues of said modified amidotransferase are substituted at positions equivalent to the amino acid positions in the Bacillus amidotransferase selected from the group consisting of 282, 283, 307, and 347 of SEQ ID NO: I with amino acids not native to said position or positions, wherein said equivalent positions are identified based on comparison of amino acid sequences of the native amidotransferase and Bacillus glutamine PRPP amidotransferase, and wherein said modified amidotransferase is less sensitive to end-product inhibition than is the native amidotransferase.  
     
     
         6 . The method of  claim 5  wherein the native amidotransferase is a native  E. coli  glutamine PRPP amidotransferase and the modified amidotransferase is a modified  E. coli  glutamine PRPP amidotransferase.  
     
     
         7 . The method of  claim 6  wherein the amino acid substitution reduces synergistic binding of adenine and guanine nucleotides to the amidotransferase.  
     
     
         8 . A method for purine nucleotide biosynthesis, said method comprising the step of culturing a host cell in a medium suitable for purine nucleotide biosynthesis, said host cell transformed with a nucleic acid sequence encoding a modified  E. Coli  glutamine PRPP amidotransferase, wherein the amino acid sequence of said modified  E. Coli  amidotransferase differs from the amino acid sequence of a native  E coli  glutamine PRPP amidotransferase in that one or more amino acid residues of said modified amidotransferase are substituted at positions equivalent to the amino acid positions in the Bacillus amidotransferase selected from the group consisting of 282, 283, 307, and 347 of SEQ ID NO:1 with amino acids not native to said position or positions, wherein said equivalent positions are identified based on comparison of amino acid sequences of Bacillus and  E. Coli  glutamine PRPP amidotransferases, and wherein said modified  E. coli  amidotransferase is less sensitive to end-product inhibition than is the native  E. coli  glutamine PRPP amidotransferase.  
     
     
         9 . The method of  claim 8  wherein the nucleic acid sequence encoding the glutamine PRPP amidotransferase is operably linked to a constitutive promoter.