Novel enoyl reductases and methods of use thereof

Abstract

The present provides structurally related enzymes that act as enoyl reductases. These enoyl reductases share a common amino acid consensus sequence, and bind a flavin cofactor. One particular enoyl reductase provided, FabK, catalyzes the identical reaction as the NADH-dependent enoyl-ACP reductase, FabI. Nucleic acids encoding the enoyl reductases, the enoyl reductases, and anitbodies for the enoyl reductases are also included. Methods are also provides for identifying agents that can act to prevent and/or treat bacterial infections.

1 . An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:45, wherein said polypeptide acts enzymatically as an enoyl reductase and binds a flavin prosthetic group.  
     
     
         2 . The isolated nucleic acid of  claim 1  wherein the polypeptide is a bacterial enzyme or an active fragment of the bacterial enzyme.  
     
     
         3 . The isolated nucleic acid of  claim 2  wherein the bacterial enzyme has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:2 comprising a conservative amino acid substitution, SEQ ID NO:4, SEQ ID NO:4 comprising a conservative amino acid substitution, SEQ ID NO:6, SEQ ID NO:6 comprising a conservative amino acid substitution, SEQ ID NO:10, SEQ ID NO:10 comprising a conservative amino acid substitution, SEQ ID NO:12, SEQ ID NO:12 comprising a conservative amino acid substitution, SEQ ID NO:14, SEQ ID NO:14 comprising a conservative amino acid substitution, SEQ ID NO:16, SEQ ID NO:16 comprising a conservative amino acid substitution, SEQ ID NO:18, SEQ ID NO:18 comprising a conservative amino acid substitution, SEQ ID NO:20, and SEQ ID NO:20 comprising a conservative amino acid substitution.  
     
     
         4 . The isolated nucleic acid of  claim 3  comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3; SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.  
     
     
         5 . An isolated nucleic acid that hybridizes under standard hybridization conditions to a cDNA comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3; SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.  
     
     
         6 . A recombinant DNA molecule that consists of the isolated nucleic acid of  claim 1  and a heterologous nucleotide sequence.  
     
     
         7 . A recombinant DNA molecule that is operatively linked to an expression control sequence, wherein the recombinant DNA comprises the isolated nucleic acid of  claim 1 .  
     
     
         8 . An expression vector containing the recombinant DNA of  claim 6 .  
     
     
         9 . A cell comprising the expression vector of  claim 7 .  
     
     
         10 . A method of expressing a recombinant polypeptide in the cell of  claim 8  comprising culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the polypeptide by the cell, wherein said recombinant polypeptide comprises the amino acid sequence of SEQ ID NO:45, can bind a flavin prosthetic group and can act enzymatically as an enoyl reductase.  
     
     
         11 . The method of  claim 9  further comprising the step of purifying the recombinant polypeptide.  
     
     
         12 . (canceled)  
     
     
         13 . A nucleic acid comprising a polypeptide that has at least 80% identity with a bacterial enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20; wherein said polypeptide binds a flavin prosthetic group and has enoyl reductase activity.  
     
     
         14 . A nucleic acid comprising a polypeptide that comprises at least 12 consecutive amino acids of a bacterial enzyme that has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ ID NO:20; wherein said polypeptide binds a flavin prosthetic group and has enoyl reductase activity.  
     
     
         15 - 25 . (canceled)  
     
     
         26 . An antibody to the antigenic fragment of a peptide that has an amino acid sequence selected from the group consisting of SEQ ID NO:46 or SEQ ID NO:46 comprising a conservative amino acid substitution.  
     
     
         27 . The antibody of  claim 26  that is a monoclonal antibody.  
     
     
         28 . The antibody of  claim 27  that is a chimeric antibody.  
     
     
         29 . An immortal cell line that produces a monoclonal antibody of  claim 27 .  
     
     
         30 . An antibody to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:45, wherein said polypeptide acts enzymatically as an enoyl reductase and binds a flavin prosthetic group; and wherein said polypeptide is not a yeast enzyme.  
     
     
         31 . A method for identifying an agent that can modulate the enzymatic activity of an enoyl reductase comprising: 
 (a) measuring the enzymatic activity of an enoyl reductase or active fragment thereof in the presence and absence of a compound; wherein said enoyl reductase comprises the amino acid sequence of SEQ ID NO:45 and a flavin prosthetic group, or the amino acid sequence of SEQ ID NO:57; and    (b) identifying the compound as an agent that modulates the enzymatic activity of an enoyl reductase when the enzymatic activity measured in step (a) is different in the presence of the compound relative to in the absence of the compound.    
     
     
         32 . The method of  claim 31  wherein the enzymatic activity is lower in the presence of the compound relative to in the absence of the compound, and wherein the compound is identified as an inhibitor.  
     
     
         33 . The method of  claim 31  wherein the enoyl reductase has the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:2 comprising a conservative amino acid substitution, SEQ ID NO:4, SEQ ID NO:4 comprising a conservative amino acid substitution, SEQ ID NO:6, SEQ ID NO:6 comprising a conservative amino acid substitution, SEQ ID NO:10, SEQ ID NO:10 comprising a conservative amino acid substitution, SEQ ID NO:12, SEQ ID NO:12 comprising a conservative amino acid substitution, SEQ ID NO:14, SEQ ID NO:14 comprising a conservative amino acid substitution, SEQ ID NO:16, SEQ ID NO:16 comprising a conservative amino acid substitution, SEQ ID NO:18, SEQ ID NO:18 comprising a conservative amino acid substitution, SEQ ID NO:20, SEQ ID NO:20 comprising a conservative amino acid substitution, SEQ ID NO:52, SEQ ID NO:52 comprising a conservative amino acid substitution, SEQ ID NO:54, SEQ ID NO:54 comprising a conservative amino acid substitution, SEQ ID NO:56, SEQ ID NO:56 comprising a conservative amino acid substitution, SEQ ID NO:50, and SEQ ID NO:50 comprising a conservative amino acid substitution.  
     
     
         34 . A method for identifying an agent that can bind to an enoyl reductase comprising: 
 (a) contacting an enoyl reductase or active fragment thereof with a compound; wherein said enoyl reductase comprises the amino acid sequence of SEQ ID NO:45 and a flavin prosthetic group or the amino acid sequence of SEQ ID NO:57; and    (b) determining if the compound binds to enoyl reductase; wherein a compound is identified as an agent that binds the enoyl reductase when the compound binds to the enoyl reductase.    
     
     
         35 . The method of  claim 34  wherein the enoyl reductase has the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:2 comprising a conservative amino acid substitution, SEQ ID NO:4, SEQ ID NO:4 comprising a conservative amino acid substitution, SEQ ID NO:6, SEQ ID NO:6 comprising a conservative amino acid substitution, SEQ ID NO:10, SEQ ID NO:10 comprising a conservative amino acid substitution, SEQ ID NO:12, SEQ ID NO:12 comprising a conservative amino acid substitution, SEQ ID NO:14, SEQ ID NO:14 comprising a conservative amino acid substitution, SEQ ID NO:16, SEQ ID NO:16 comprising a conservative amino acid substitution, SEQ ID NO:18, SEQ ID NO:18 comprising a conservative amino acid substitution, SEQ ID NO:20, SEQ ID NO:20 comprising a conservative amino acid substitution, SEQ ID NO:52, SEQ ID NO:52 comprising a conservative amino acid substitution, SEQ ID NO:54, SEQ ID NO:54 comprising a conservative amino acid substitution, SEQ ID NO:56, SEQ ID NO:56 comprising a conservative amino acid substitution, SEQ ID NO:50, and SEQ ID NO:50 comprising a conservative amino acid substitution.  
     
     
         36 . A method for identifying a drug that inhibits bacterial growth comprising: 
 (a) administering the agent of claims  31  to a bacterial cell;    (b) determining the growth of the cell; wherein an agent that inhibits the growth of the cell relative to the growth in the absence of the agent is identified as a drug that inhibits bacterial growth.    
     
     
         37 . A pharmaceutical composition comprising the drug of  claim 36  and a pharmaceutically acceptable carrier.  
     
     
         38 . An isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:57, wherein said polypeptide acts enzymatically as an enoyl reductase.  
     
     
         39 . The isolated nucleic acid of  claim 38  wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:52 comprising a conservative amino acid substitution.  
     
     
         40 - 43 . (canceled)