RECOMBINANT BIOTIN CARBOXYLASE DOMAINS FOR IDENTIFICATION OF ACETYL CoA CARBOXYLASE INHIBITORS

Abstract

A peptide comprising an Acetyl CoA carboxylase (ACCase) having a deleted biotin binding domain, having a deleted carboxy transferase domain, and having a functional biotin carboxylase (BC) domain is described. A nucleic acid that encodes the peptide described above and a recombinant host cell that contains the nucleic acid and expresses the encoded peptide is also described. A method of identifying Acetyl CoA carboxylase inhibitors, fungicides, herbicides and pharmaceuticals is also described herein.

1 . A peptide comprising an Acetyl CoA carboxylase (ACCase) having a deleted biotin binding domain, having a deleted carboxy transferase domain, and having a functional biotin carboxylase domain. 
     
     
         2 . The peptide according to  claim 1 , wherein said ACCase is selected from the group consisting of mammal, insect, yeast, Ascomycota, Basidiomycota, and Oomycota ACCase. 
     
     
         3 . The peptide according to  claim 1 , wherein said carboxylase is  Ustilago maydis  carboxylase. 
     
     
         4 . The peptide according to  claim 1 , wherein said carboxylase is  Phytopthora infestans  carboxylase. 
     
     
         5 . The peptide according to  claim 1 , wherein said carboxylase is  Magnaporthe grisea  carboxylase. 
     
     
         6 . The peptide according to  claim 1 , wherein said carboxylase is  Saccaromyces cerevisiae  carboxylase. 
     
     
         7 . The peptide according to  claim 1 , wherein said carboxylase is human carboxylase. 
     
     
         8 . The peptide according to  claim 1  having the amino acid sequence given in SEQ ID NO: 2. 
     
     
         9 . The peptide according to  claim 1  selected from the group consisting of peptides having an amino acid sequence as given in SEQ ID NO; 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17 through SEQ ID NO: 71. 
     
     
         10 . The peptide according to  claim 1 , wherein said peptide is a monomer. 
     
     
         11 . The peptide according to  claim 1 , wherein said peptide binds to soraphen. 
     
     
         12 . The peptide according to  claim 1 , wherein said peptide binds to soraphen and has a soraphen dissociation constant of from 10 −7  to 10 −14  M. 
     
     
         13 . A composition comprising:
 (a) an aqueous carrier solution; and   (b) the peptide of  claim 1  solubilized in said aqueous carrier solution; with   said peptide included in said composition in an amount of from 0.001 nanograms to 20 milligrams per milliliter of aqueous carrier solution;   said peptide having a soraphen dissociation constant in said composition of from 10 −7  to 10 −14  M; and   said composition having a pH of from 5 through 9.   
     
     
         14 . A nucleic acid that encodes a peptide according to  claim 1 . 
     
     
         15 . A recombinant host cell that contains a nucleic acid according to  claim 14  and expresses the encoded peptide. 
     
     
         16 . A method of identifying Acetyl CoA carboxylase inhibitors or activators, comprising:
 a) combining a peptide according to  claim 1  and a compound to be tested for the ability to bind to said biotin carboxylase domain, under conditions that permit binding to said biotin carboxylase domain;   b) determining whether or not said compound binds to said biotin carboxylase domain, the presence of binding indicating said compound is or may be an Acetyl CoA carboxylase inhibitor or activator.   
     
     
         17 . The method of  claim 16 , further comprising the steps of:
 c) employing a compound identified as binding in step (b) in an assay to detect inhibition or enhancement of Acetyl CoA carboxylase activity; and   d) selecting a compound identified in step (c) that inhibits or activates Acetyl CoA carboxylase activity.   
     
     
         18 . A method of identifying fungicides, comprising:
 a) combining a peptide according to  claim 1  and a compound to be tested for the ability to bind to said biotin carboxylase domain, under conditions that permit binding to said biotin carboxylase domain;   b) determining whether or not said compound binds to said biotin carboxylase domain, the presence of binding indicating said compound is or may be a fungicide;   c) employing a compound identified as binding in step (b) in an assay to detect inhibition of Acetyl CoA carboxylase activity; and   d) selecting a compound identified in step (c) that inhibits Acetyl CoA carboxylase activity.   
     
     
         19 . A kit comprising:
 (a) a first peptide of  claim 1 ; in combination with   (b) a second peptide of  claim 1 ,   wherein said first and second peptides are from different species.   
     
     
         20 . A kit of  claim 19 , wherein said first peptide is a non-mammalian peptide and said second peptide is a mammalian peptide. 
     
     
         21 . A kit comprising:
 (a) a first peptide of  claim 1 ; in combination with   (b) a second peptide comprising an ACCase having a deleted biotin binding domain, having a deleted carboxy transferase domain, and having a non-functional biotin-carboxylase domain;   wherein said first and second peptide are from the same species.   
     
     
         22 . A kit of  claim 19 , wherein said first and second peptide are both  S. cerivasae  peptides.