A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates.
Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli. The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F. Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors. Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F. The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993). The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.
Zapf
J W
JW
Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Hoch
J A
JA
Whiteley
J M
JM
D001224:Aspartic Acid; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015290:Second Messenger Systems; D015398:Signal Transduction; D013171:Spores, Bacterial; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002413:Cations, Divalent; D017931:DNA Primers; D011994:Recombinant Proteins; C080951:Spo0F protein, Bacillus subtilis; D001224:Aspartic Acid; D010770:Phosphotransferases; D008274:Magnesium