Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cell-wall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs. To explore the distinguishing characteristics of the five Mtb Rpfs, we determined the crystal structure of the RpfE catalytic domain. The protein adopts the characteristic Rpf fold, but the catalytic cleft is narrower compared to Mtb RpfB. Also in contrast to RpfB, in which the substrate-binding surfaces are negatively charged, the corresponding RpfE catalytic pocket and predicted peptide-binding sites are more positively charged at neutral pH. The complete reversal of the electrostatic potential of the substrate-binding site suggests that the different Rpfs function optimally at different pHs or most efficiently hydrolyze different micro-domains of PG. These studies provide insights into the molecular determinants of the evolution of functional specialization in Rpfs.