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

Abstract

<p>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.</p>

Topics

• impedance spectroscopy
• activation
• High-performance liquid chromatography
• particle-induced X-ray emission spectroscopy