Identification and characterization of novel proline racemases and hydroxyproline-2-epimerases, uses thereof, and methods of identifying proline racemases and hydroxyproline-2-epimerases

A method of identifying a target amino acid sequence as a putative proline racemase, wherein the method comprises:

• (A) providing an amino acid sequence database;
• (B) performing a computer assisted search of the database to compare a known amino acid sequence of a proline racemase with sequences in the database;
• (C) identifying a target amino acid sequence in the database having at least about 25% homology to the known proline racemase amino acid sequence;
• wherein the target sequence is a putative proline racemase if:
• (1) the target sequence has the motif MIII* [SEQ ID NO:
• 4];
• (2) the target sequence has the motif MCGH;
• (3) the target sequence has two catalytic Cys residues corresponding to Cys160 and Cys330 (or Cys130 and Cys300 when the signal sequence is not present) of Trypanosoma cruzi racemase (TcPRAC) [SEQ ID NO:
• 30];
• and
• (4) the target sequence has a phenylalanine residue corresponding to Phe132 (or Phe102 when the signal sequence is not present)(R1) of TcPRAC;
• and
• (5) the target sequence has a Cys or Leu (R2) residue corresponding to Cys300 (or Cys270 when the signal sequence is not present) of TcPRAC.

A method of identifying a target amino acid sequence as a putative epimerase, wherein the method comprises:

• (A) providing an amino acid sequence database;
• (B) performing a computer assisted search of the database to compare a known amino acid sequence of a proline racemase with sequences in the database;
• (C) identifying a target amino acid sequence in the database having at least about 25% homology to the known proline racemase amino acid sequence;
• wherein the target sequence is a putative epimerase if:
• (1) the target sequence has the motif MIII* [SEQ ID NO:
• 4];
• (2) the target sequence has the motif MCGH;
• (3) the target sequence has two catalytic Cys residues corresponding to Cys160 and Cys330 (or Cys130 and Cys300 if the signal sequence is not present) of Trypanosoma cruzi racemase (TcPRAC) [SEQ ID NO:
• 30];
• and
• (4) the target sequence has a Ser or Val residue corresponding to Phe132 (or Phe102 when the signal sequence is not present) (R1) of TcPRAC;
• and
• (5) the target sequence has a H is residue corresponding to Cys300 (or Cys270 when the signal sequence is not present) of TcPRAC;
• and
• (6) the target sequence has the R3 motif [SEQ ID NO:
• 130], which is absent from TcPRAC.

A method for the catalyzed epimerization of OH-L-Pro and OH-D-Pro of 4-hydroxyproline, wherein the method comprises:

• (A) providing at least one epimerase selected from:
• EF495341 (PaHyPRE, P. aeruginosa PAK) [SEQ ID NO:
• 131],
• EF495342 (BmHyPRE, B. melitensis 16M) [SEQ ID NO:
• 133],
• EF495343 (BsHyPRE, B. suis 1330) [SEQ ID NO:
• 135],
• EF495344 (BaHyPRE, B. abortus 544) [SEQ ID NO 137], and
• EF495345 (BpHyPRE, B. pseudomallei K96243) [SEQ ID NO:
• 139];
• and
• (B) reacting the epimerase with 4-hydroxyproline in the presence of a buffer and at a pH for OH-L/D-Pro epimerization.

A method of detecting a substrate for a biologically active protein, wherein the method comprises:

• (A) providing a composition suspected of containing the substrate;
• (B) contacting the composition with EF495346 (CdPRAC, C. difficile VPI10463) [SEQ ID NO:
• 141] or EF175213 (TVPRAC, T. vivax [SEQ ID NO:143];
• and
• (C) assaying the resulting mixture for L-Pro racemization, D-Pro racemization, or both.

A method of detecting a substrate for a biologically active protein, wherein the method comprises:

• (A) providing a composition suspected of containing the substrate;
• (B) contacting the composition with at least one epimerase selected from:
• EF495341 (PaHyPRE, P. aeruginosa PAK) [SEQ ID NO:
• 131],
• EF495342 (BmHyPRE, B. melitensis 16M) [SEQ ID NO:
• 133],
• EF495343 (BsHyPRE, B. suis 1330) [SEQ ID NO:
• 135],
• EF495344 (BaHyPRE, B. abortus 544) [SEQ ID NO:
• 137], and
• EF495345 (BpHyPRE, B. pseudomallei K96243) [SEQ ID NO:
• 139];
• and
• (C) assaying the resulting mixture for OH-L-Pro epimerization, OH-D-Pro epimerization, or both.

A method for detecting an inhibitor of a biologically active protein, wherein the method comprises:

• (A) providing a composition suspected of containing an inhibitor of the biologically active protein;
• (B) contacting the composition with EF495346 (CdPRAC, C. difficile VPI10463) [SEQ ID NO:141] or EF175213 (TVPRAC, T. vivax) [SEQ ID NO:143];
• and
• (C) assaying the resulting mixture for the inhibition of L-Pro racemization, D-Pro racemization, or both.

A method for detecting an inhibitor for a biologically active protein, wherein the method comprises:

• (A) providing a composition suspected of containing an inhibitor of the biologically active protein;
• B) contacting the composition with at least one epimerase selected from:
• EF495341 (PaHyPRE, P. aeruginosa PAK) [SEQ ID NO:
• 131],
• EF495342 (BmHyPRE, B. melitensis 16M) [SEQ ID NO:
• 133],
• EF495343 (BsHyPRE, B. suis 1330) [SEQ ID NO:
• 135],
• EF495344 (BaHyPRE, B. abortus 544) [SEQ ID NO:
• 137], and
• EF495345 (BpHyPRE, B. pseudomallei K96243) [SEQ ID NO:
• 139];
• and
• (C) assaying the resulting mixture for the inhibition of OH-L-Pro epimerization, OH-D-Pro epimerization, or both.

A hydroxyproline epimerase comprising an amino acid sequence selected from SEQ ID NO:

• 131, SEQ ID NO:
• 133, SEQ ID NO:
• 135, SEQ ID NO:
• 137, and SEQ ID NO:
• 139.

A method for treating a patient infected with Clostridium difficile or Tripanosoma vivax, comprising administering to the patient an active molecule capable of inhibiting the proline racemase activity of a protein comprising the amino acid sequence of SEQ ID NO:

• 141 or SEQ ID NO:
• 143, wherein said method has an effect chosen from:
• (a) inhibiting the growth of the Clostridium difficile or Tripanosoma vivax;
• (b) preventing the Clostridium difficile or Tripanosoma vivax from evading host cell immunity;
• and
• (c) preventing mitogen-induced proliferation of resting lymphocytes.

A method for treating a patient infected with Pseudomonas aeruginosa, Burkholderia pseudomallei, Brucella abortus, Brucella melitensis, or Brucella suis, comprising administering to the patient an active molecule capable of inhibiting the hydroxyproline epimerase activity of a protein comprising the amino acid sequence of SEQ ID NO:

• 131, SEQ ID NO:
• 133, SEQ ID NO:
• 135, SEQ ID NO:
• 137, or SEQ ID NO:
• 139, respectively, wherein said method has an effect chosen from:
• (a) inhibiting the growth of Pseudomonas aeruqinosa, Burkholderia pseudomallei, Brucella abortus, Brucella melitensis, or Brucella suis;
• (b) preventing Pseudomonas aeruqinosa, Burkholderia pseudomallei, Brucella abortus, Brucella melitensis, or Brucella suis from evading host cell immunity;
• and
• (c) preventing mitogen-induced proliferation of resting lymphocytes.

A method for stimulating a protective immune response against Clostridium difficile, Tripanosoma vivax, Pseudomonas aeruginosa, Burkholderia pseudomallei, Brucella abortus, Brucella melitensis, or Brucella suis in a patient, comprising administering to the patient a purified mitogen comprising an amino acid sequence selected from SEQ ID NO:

• 141, SEQ ID NO:
• 143, SEQ ID NO:
• 131, SEQ ID NO 133, SEQ ID NO:
• 135, SEQ ID NO:
• 137, and SEQ ID NO:
• 139, respectively, or a portion thereof, wherein the purified mitogen or portion thereof is capable of inducing an immune response in vivo.

A method for stimulating a protective immune response against Clostridium difficile, Tripanosoma vivax, Pseudomonas aeruginosa, Burkholderia pseudomallei, Brucella abortus, Brucella melitensis, or Brucella suis in a patient, comprising administering to the patient a purified nucleic acid encoding a polypeptide comprising an amino acid sequence selected from SEQ ID NO:

• 141, SEQ ID NO:
• 143, SEQ ID NO:
• 131, SEQ ID NO 133, SEQ ID NO:
• 135, SEQ ID NO:
• 137, and SEQ ID NO:
• 139, respectively, or a portion thereof, wherein the polypeptide or portion thereof encoded by the nucleic acid is capable of inducing an immune response in vivo.

A purified antibody directed against an amino acid sequence selected from SEQ ID NO:

• 141, SEQ ID NO:
• 143, SEQ ID NO:
• 131, SEQ ID NO 133 SEQ ID NO:
• 135, SEQ ID NO:
• 137 and SEQ ID NO:
• 139 or an antigenic fragment thereof.