Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T(35)IXXSQ ELDCLI(46) residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0A approximately P was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr(35) and Cys(44) did not seem to have any critical functional or structural role. We propose that Asp(43) of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226 ).