Methods and compositions for determining enzymatic activity

Abstract

The present invention comprises crystalline polyketide synthases, isolated non-native polyketide synthases having the structural coordinates of said crystalline polyketide synthases, and nucleic acids encoding such non-native polyketide synthases. Also disclosed are methods of predicting the activity and/or substrated specificity of putative polyketide synthase, methods of identifying potential polyketide synthase substrates, and methods of identifying potential polyketide synthase inhibitors.

1 . A nucleic acid encoding a polyketide synthase comprising at least fourteen active site α-carbons having the structural coordinates of Table 1. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the amino acid located at position 164 is alanine or serine. 
     
     
         3 . The nucleic acid of  claim 1 , wherein the amino acid located at position 303 is alanine, asparagine, glutamine, aspartic acid, or threonine. 
     
     
         4 . The nucleic acid of  claim 1 , wherein the amino acid located at position 336 is a lysine, alanine, aspartic acid, glutamine, or histidine. 
     
     
         5 . The nucleic acid of  claim 1 , wherein the amino acid located at position 215 is serine, tyrosine, or tryptophan. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the polyketide synthase has atomic coordinates as set forth in PUB Accession Nos: 1B15, 1BQ6, 1CML, 1CHW, 1CGK, 1CGZ, 1D6F, 1D61, or 1D6H. 
     
     
         7 . An isolated polyketide synthase encoded by the nucleic acid of  claim 1 . 
     
     
         8 . An isolated polyketide synthase encoded by the nucleic acid of  claim 2 . 
     
     
         9 . An isolated polyketide synthase encoded by the nucleic acid of  claim 3 . 
     
     
         10 . An isolated polyketide synthase encoded by the nucleic acid of  claim 4 . 
     
     
         11 . An isolated polyketide synthase encoded by the nucleic acid of  claim 5 . 
     
     
         12 . An isolated polyketide synthase encoded by the nucleic acid of  claim 6 . 
     
     
         13 . A method of identifying a potential substrate of a polyketide synthase according to  claim 7 , said method comprising:
 (a) defining the active site of said polyketide synthase based on a plurality of atomic coordinates of said polyketide synthase,   (b) identifying a potential substrate that fits the active site of (a) with the polyketide synthase, and   (c) contacting the polyketide synthase with the potential substrate and determining its activity thereon.   
     
     
         14 . The method of  claim 13 , wherein the polyketide synthase is chalcone synthase, stilbene synthase, or pyrone synthase. 
     
     
         15 . The method of  claim 13 , wherein the polyketide synthase is a mutant of a known polyketide synthase selected from chalcone synthase, stilbene synthase, or pyrone synthase. 
     
     
         16 . The method of  claim 13 , wherein the plurality of atomic coordinates are as set forth in PDB Accession Nos: 1BI5, 1BQ6, 1CML, 1CHW, 1CGK, 1CGZ, 1D6F, 1D6I, 1D6H, or portions thereof. 
     
     
         17 . A method of identifying a potential inhibitor of a polyketide synthase according to  claim 7 , said method comprising:
 (a) defining the active site of said polyketide synthase based on a plurality of atomic coordinates of said polyketide synthase,   (b) contacting a potential compound that fits the active site of (a) with the polyketide synthase in the presence of a substrate, and   (c) determining the ability of said compound to inhibit the activity of said polyketide synthase on said substrate.   
     
     
         18 . The method of  claim 17 , wherein the polyketide synthase is chalcone synthase, stilbene synthase, or pyrone synthase. 
     
     
         19 . The method of  claim 17 , wherein the polyketide synthase is a mutant of a known polyketide synthase selected from chalcone synthase, stilbene synthase, or pyrone synthase. 
     
     
         20 . The method of  claim 17 , wherein the plurality of atomic coordinates are as set forth in PDB Accession Nos: 1BI5, 1BQ6, 1CML, 1CHW, 1CGK, 1CGZ, 1D6F, 1D6I, 1D6H, or portions thereof.