The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization
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abstract
Characterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. Here, we report a study ofBacillus subtilisYdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of theydiBgene yielded a mutant with much reduced growth rate compared to the wild-type strain.In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed aKmof about 60 μM for ATP and aVmaxof about 10 nmol min−1(mg protein)−1(kcat∼10 h−1). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain forydiB, showing that the ATPase activity of YdiB is required for thein vivofunction of the protein. Oligomerization was also observed with the purified YjeE fromEscherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in aB. subtilisextract, or when stabilized by formaldehyde cross-linking for YjeE fromE. coli, suggesting that oligomerization might regulate the function of this new class of proteinsin vivo.
