The ATPase activity of an 'essential' Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization.
Characterization of 'unknown' proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene 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 a K(m) of about 60 microM for ATP and a V(max) of about 10 nmol min(-1) (mg protein)(-1) (k(cat) approximately 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 for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia 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 a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.
Karst
Johanna C
JC
Institut de Biologie Structurale, UMR 5075 Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
Foucher
Anne-Emmanuelle
AE
Institut de Biologie Structurale, UMR 5075 Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
Campbell
Tracey L
TL
Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.
Di Guilmi
Anne-Marie
AM
Institut de Biologie Structurale, UMR 5075 Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
Stroebel
David
D
Institut de Biologie Structurale, UMR 5075 Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
Mangat
Chand S
CS
Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.
Brown
Eric D
ED
Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada.
Jault
Jean-Michel
JM
Institut de Biologie Structurale, UMR 5075 Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France.
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D055503:Protein Multimerization
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases