Isolation and Identification of Protein l-Isoaspartate-O-Methyltransferase (PIMT) Interacting Proteins in Salmonella Typhimurium

Protein l-isoaspartate-O-methyltransferase (PIMT) plays an important role in restoration of covalently damaged Asn/Asp residues. It repairs the racemized forms of these amino acids in protein by forming a labile isoAsp methyl ester which readily converts back to the succinimide intermediate. Spontaneous hydrolysis of the intermediate further restores a minor portion to the normal Asp residues. While significant numbers of PIMT targets have been identified in eukaryotes, very few are documented from prokaryotes. Temperature (42 °C) induced elevation in PIMT expression level has been recently shown in a poultry isolate of Salmonella Typhimurium (ST). The enzyme was also found to be crucial for survival, virulence and colonization of ST in poultry. In the present study, co-immunoprecipitation (Co-IP) approach was used (for isolation) followed by LC–MS analysis to identify the PIMT interacting proteins of ST. Four different proteins were identified among which cytochrome C biogenesis protein A (CcmA) was further expressed in recombinant form and analysed for interaction with recombinant PIMT (rPIMT) by microtiter plate assay. Additionally, the findings were supported by alterations in secondary structure of the proteins upon co-incubation.