A Phosphotransferase Activity of theBacillus subtilisSporulation Protein Spo0F That Employs Phosphoramidate Substrates

Abstract

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.

The Acetate Switch

“…Other small phosphorylated compounds (carbamyl phosphate, phosphoramidate, monophosphoimidazole, and diphosphoimidazole) can perform a similar function, although no evidence exists that they perform this function in vivo. In contrast, neither ATP, PEP, nor creatine phosphate can donate their phosphoryl group to any RR (58,115,127,190,279,296,297,376,383,411,486). Each RR responds uniquely with respect to phosphoryl donor preference and its rate of autophosphorylation.…”

1H,15N, and13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of thebacillus subtilisresponse regulator, SpoOF, determined by heteronuclear high-resolution NMR

“…SpoOF requires magnesium for phosphorylation by either KinA, KinB, or chemical phospho donors (Hoch, 1993b;Zapf et al, 1995). To characterize the affinity and structural consequences of magnesium binding, chemical shift changes of backbone amide proton resonances were followed by 'H,"N HSQC or ' H , "N HSMQC spectra as magnesium concentration was increased.…”

“…Binding to the kinases, KinA or KinB, may alter the SpoOF magnesium affinity and ensure that the protein becomes phosphorylated only in response to the appropriate signal and by the correct kinase. Secondly, because magnesium is required for the autodephosphorylation in SpoOF (Zapf et al, 1995), the low affinity for magnesium may explain the long lifetimes observed for SpoOF-P in vitro. The observed tl,* for SpoOF in vitro is 12 h, three orders of magnitude longer than lifetimes observed for CheY (Lukat et al, 1990).…”

“…More recently, expression levels have been enhanced to give a yield of 15 mg of purified protein per liter of rich media using PET expression vectors (Novagen, Madison, Wisconsin). The spoOF open reading frame was PCR amplified from pKKOF (Zapf et al, 1995) using oligonucleotides complementary to the terminal portions of the spoOF gene and contained artificial Nde 1 and BamH I sites. The spoOF gene was subcloned into the vector pET20b with Nde I and BamH 1.…”

“…SpoOF was purified in a three-step procedure entailing DEAETrisacryl, Bio-Gel HTP hydroxylapatite, and gel-filtration column chromatography as described by Zapf et al (1995). Addition of the hydroxylapatite column chromatography step served to remove a 30-kDa protein contaminant observed in Protocol 1.…”