A phosphotransferase activity of the Bacillus subtilis sporulation protein SpoOF that employs phosphoramidate substrates

Transient phosphorylation at an aspartate residue on the SpoOF protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli. The response regulator SpoOF similar to P is stable to hydrolysis (t(1/2) > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated farms of SpoOF. Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate SpoOF, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors. Rapid hydrolysis of SpoOF similar to P generated with phosphoramidate by proteins downstream in the phosphorelay (SpoOB and SpoOA) is consistent with phosphorylation at the active site of SpoOF. The initial rate of SpoOF similar to P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as SpoOF, function as phosphoryl transferase enzymes (McCleary et al., 1993). The results establish that SpoOF functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.