Shared Catalysis in Virus Entry and Bacterial Cell Wall Depolymerization

Daniel N. Cohen, Yuk Y. Sham, Greg D. Haugstad, Ye Xiang, Michael G. Rossmann, Dwight L. Anderson, David L. Popham

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26 Scopus citations


Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and a structural pattern of β-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage φ{symbol}29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a d,d-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn2+ metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn2+ binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn2+ binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action.

Original languageEnglish (US)
Pages (from-to)607-618
Number of pages12
JournalJournal of Molecular Biology
Issue number3
StatePublished - Apr 3 2009

Bibliographical note

Funding Information:
D.N.C., Y.Y.S., G.D.H., D.L.A., and D.L.P. designed the experiments, while D.N.C., Y.Y.S., G.D.H., and D.L.P. performed them. D.N.C., Y.Y.S., G.D.H., D.L.A., and D.L.P. analyzed the data. Y.X. and M.G.R. provided the gp13 plasmid and the purified N-terminal protein used in this study. D.N.C., Y.Y.S., D.L.A., and D.L.P. wrote the article. This work was supported by the National Institutes of Health through grant DE003606 (to D.L.A.) from the National Institute of Dental and Craniofacial Research and grant GM056695 (to D.L.P.) from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Dental and Craniofacial Research, the National Institute of General Medical Sciences, or the National Institutes of Health. We thank Dr. John Lipscomb for critical reading of previous versions of the manuscript and Dr. Matthias Bochtler for helpful discussion. We also thank Jessica McElligott for technical assistance and the University of Minnesota Supercomputing Institute for providing the computational resource for the molecular modeling study as well as the Characterization Facility (which receives partial support from the National Science Foundation through the National Nanotechnology Infrastructure Network program) for providing the ion beam analysis resources.


  • autolysin
  • bacteriophage
  • gp13
  • metallopeptidase
  • φ{symbol}29


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