Anand S, et al. - In view of the observation that Eis (Enhanced Intracellular Survival), an important aminoglycoside N-acetyltransferase enzyme contributing to kanamycin resistance in Mtb clinical isolates, from M. tuberculosis (RvEis) and M. smegmatis (MsEis) have 58% identical and 69% similar amino acid sequences and acetylate aminoglycosides at multiple amines, a comparative analysis of structural stability and aminoglycoside acetyltransferase activity of RvEis and MsEis proteins was performed. Observations revealed a three-state unfolding induced by heat or chemical denaturants and involvement of self-association of partially unfolded oligomers to form high molecular weight soluble aggregates in MsEis, not in RvEis. At lower concentrations of GdmCl and urea, MsEis vs RvEis is highly susceptible to chemical denaturants and unfolds completely. Collectively, findings suggest different modes of unfolding and differential structural stability and activity among homologous proteins from pathogenic and nonpathogenic mycobacteria, despite a high similarity in sequences and oligomeric organization.