Acyl glucosaminyl inositol amidase family and methods of use

Abstract

The present invention provides a family of bacterial acyl glucosaminylinositol amidases with amidase activity against S-conjugate amides, particularly mycothiol-derived S-conjugate amides. The invention amidases are characterized by a highly conserved 20 amino acid N-terminal region and four highly conserved histidine-containing regions and by having amidase activity, particularly amide hydrolase activity. The invention further provides methods for using the invention amidases in drug screening assays to determine compounds with antibiotic activity or compounds that inhibit activity or production of endogenous acyl glucosaminyl inositol amidase in bacteria. The invention further provides methods for detoxifying a toxic substance by contacting the toxic substance with an invention amidase, for example, by expression of the amidase under environmental conditions in a bacterium.

That which is claimed is:  
     
         1 . A purified acyl glucosaminyl inositol amidase, characterized as having: 
 a) an N-terminal region with an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2,    b) four domains of conservation, wherein three of the domains contain conserved histidine residues, and    c) amidase activity against acyl glucosaminyl inositol amides.    
     
     
         2 . A purified amidase of  claim 1 , wherein the amidase has amide hydrolase activity.  
     
     
         3 . A purified amidase of  claim 1 , wherein the acyl glucosaminyl inositol amide is a mycothiol-derived S-conjugate.  
     
     
         4 . A purified amidase of  claim 1 , wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.  
     
     
         5 . A purified amidase of  claim 1 , wherein the amidase is derived from an actinomycetes.  
     
     
         6 . A purified amidase of  claim 1 , wherein the amidase is a mycothiol S-conjugate amidase.  
     
     
         7 . A purified amidase of  claim 5 , wherein the amidase is derived from  M. smegmatis  and the N-terminal region has the amino acid sequence as set forth in SEQ ID NO. 2.  
     
     
         8 . A purified amidase of  claim 5 , wherein the amidase is derived from  M. tuberculosis.    
     
     
         9 . A purified amidase of  claim 5 , wherein the amidase is derived from  M. leprae.    
     
     
         10 . A purified amidase of  claim 5 , wherein the amidase is derived from  M bovis.    
     
     
         11 . A purified amidase of  claim 5 , wherein the amidase is derived from  M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum. M. chelonai, Corynebacterium diphtheria, Actinomyces israelii,  or  M. avium.    
     
     
         12 . A purified amidase of  claim 1 , wherein the amidase is derived from an antibiotic-producing bacterium.  
     
     
         13 . A purified amidase of  claim 12 , wherein the bacterium is selected from the group consisting of  Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei  and  Saccharopolyspora erythraea.    
     
     
         14 . A purified amidase of  claim 1 , wherein the amidase is encoded by a polynucleotide having a nucleic acid sequence as set forth in nucleic acid residues 34318-35184 of GenBank Accession No. gi2896719.  
     
     
         15 . A purified amidase of  claim 1 , wherein the amidase has an amino acid sequence as set forth in GenBank Accession No. CAA17198.  
     
     
         16 . A purified amidase of  claim 1 , wherein the amidase has an amino acid sequence as set forth in SEQ ID NO:1.  
     
     
         17 . A purified amidase of  claim 1 , wherein the amidase is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO:6.  
     
     
         18 . An antibody, or functional fragment thereof, that binds specifically to an amidase of  claim 1 .  
     
     
         19 . An isolated polynucleotide that encodes an amidase of  claim 1 .  
     
     
         20 . A vector containing a polynucleotide that encodes an amidase of  claim 1 .  
     
     
         21 . A cell transformed with a vector of  claim 20 .  
     
     
         22 . A method for identifying an inhibitor of acyl glucosaminyl inositol amidase, said method comprising: 
 a) contacting a candidate compound with an amidase of  claim 1  or a polynucleotide encoding the amidase in the presence of an GlcN-Ins-containing amide under suitable conditions and    b) determining the presence or absence of breakdown products of the amide indicative of amide hydrolase activity,    wherein the substantial absence of the amide hydrolase activity is indicative of a candidate compound that inhibits activity of acyl glucosaminyl inositol amidase.    
     
     
         23 . The method of  claim 22 , wherein the amidase is an acyl glucosaminyl inositol amidase.  
     
     
         24 . The method of  claim 22 , wherein the amidase is a mycothiol-derived S-conjugate amide.  
     
     
         25 . The method of  claim 22 , wherein the breakdown product is 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).  
     
     
         26 . The method of  claim 22 , wherein the breakdown product is a mercapturic acid.  
     
     
         27 . The method of  claim 22 , wherein the three domains in the amidase that contain conserved histidine residues have amino acid sequences selected from SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.  
     
     
         28 . The method of  claim 22 , wherein the amidase is produced in an actinomycete.  
     
     
         29 . The method of  claim 28 , wherein the actinomycete is  M. smegmatis  and the N-terminal region of the amidase has the amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         30 . The method of  claim 28 , wherein the actinomycete is  M. tuberculosis.    
     
     
         31 . The method of  claim 28 , wherein the actinomycete is  M. leprae.    
     
     
         32 . The method of  claim 28 , wherein the actinomycete is  M. bovis.    
     
     
         33 . The method of  claim 28 , wherein the actinomycete is  M. intracellulare, M africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii,  or  M. avium.    
     
     
         34 . A method for increasing production of antibiotic by antibiotic-producing bacteria, said method comprising: 
 contacting the antibiotic-producing bacteria with a compound that increases intracellular production by the bacteria in culture of an acyl glucosaminyl inositol amidase of  claim 1;     wherein the increase in production of the amidase increases the production of antibiotic by the bacteria by increasing resistance of the bacteria to the antibiotic.    
     
     
         35 . The method of  claim 34 , wherein the antibiotic-producing bacteria are actinomycetes.  
     
     
         36 . The method of  claim 34 , wherein candidate compound is a polypeptide, polynucleotide or small molecule.  
     
     
         37 . The method of  claim 35  wherein the compound is a polynucleotide that encodes the amidase and the actinomycetes are transformed with the polynucleotide so as to express the amidase in culture.  
     
     
         38 . The method of  claim 37 , wherein the polynucleotide comprises a nucleic acid sequence as set forth in SEQ ID NO:6.  
     
     
         39 . The method of  claim 37 , wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:1.  
     
     
         40 . The method of  claim 35 , wherein the actinomycetes are selected from the group consisting of  Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei  and  Saccharopolyspora erythraea.    
     
     
         41 . The method of  claim 34 , wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.  
     
     
         42 . The method of  claim 34 , wherein the three domains have amino acid sequences at least 80% identical to amino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.  
     
     
         43 . A method for decreasing the antibiotic-resistance of pathogenic acyl glucosaminyl inositol amidase-producing bacteria, said method comprising: 
 introducing into the bacteria an inhibitor of acyl glucosaminyl inositol amidase activity,    wherein the intracellular presence of the inhibitor decreases activity of the amidase, thereby decreasing the antibiotic-resistance of the bacteria as compared with untreated control bacteria.    
     
     
         44 . The method of  claim 43 , wherein the inhibitor inhibits intracellular production of the amidase.  
     
     
         45 . The method of  claim 43 , wherein the inhibitor inhibits intracellular amidase activity of the amidase.  
     
     
         46 . The method of  claim 43 , wherein the inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with at least 80% sequence identity to the amino acid sequence of SEQ ID NOS:2, 3, 4 or 5, and conservative variations thereof.  
     
     
         47 . The method of  claim 43 , wherein the oligonucleotide hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.  
     
     
         48 . The method of  claim 43 , wherein the bacteria are actinomycetes.  
     
     
         49 . The method of  claim 48 , wherein the bacteria are pathogenic bacteria are  M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum. M. chelonai, Corynebacterium diphtheria, Actinomyces israelii , or  M. avium.    
     
     
         50 . The method of  claim 43 , wherein the amidase is a mycothiol S-conjugate amidase.  
     
     
         51 . The method of  claim 50 , wherein conjugate partner in the S-conjugate is an electrophile.  
     
     
         52 . The method of  claim 51 , wherein the electrophile is an alkyl or alkyloid group.  
     
     
         53 . The method of  claim 43 , wherein the S-conjugate amide is a mycothiol-derived S-conjugate amide.  
     
     
         54 . The method of  claim 43 , wherein the bacteria are actinomycetes.  
     
     
         55 . The method of  claim 54 , wherein the actinomycetes are  M. smegmatis  and the N-terminal region has the amino acid sequence as set forth in SEQ ID NO. 2.  
     
     
         56 . A method for detoxifying a toxic substance comprising contacting the toxic substance with bacteria transformed with a polynucleotide that encodes an amidase of  claim 1  and expressing the amidase in order to detoxify the toxic substance.  
     
     
         57 . The method of  claim 56 , wherein the amidase is expressed under environmental conditions.  
     
     
         58 . The method of  claim 57 , wherein the environmental condition is a pollutant.  
     
     
         59 . The method of  claim 58 , wherein the pollutant includes a halogenated hydrocarbon, 1, 2 dibromoethane, 1,2 dichloroethane, perchloroethene, trichloroethene, isoprene, or vinyl chloride.  
     
     
         60 . A process for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), said method comprising: 
 contacting an N-acyl glucosaminyl inositol with an amidase of  claim 1  under suitable conditions so as to hydrolyze the amide bond therein, and  
 obtaining the GlcN-Ins.  
 
     
     
         61 . The process of claim  60 , wherein the N-acyl glucosaminyl inositol is a mycothiol S-conjugate.  
     
     
         62 . The process of claim  61 , wherein the mycothiol S-conjugate is the bimane derivative of mycothiol.  
     
     
         63 . The process of claim  61 , wherein N-acetyl glucosamine inositol is the N-acyl glucosamine inositol.  
     
     
         64 . The process of claim  63 , wherein the N-acetyl glucosamine inositol is N-acetyl- 1-D-myo-inosityl-2 amino-2 deoxy-α-D-glucopyranoside.