Shared Catalysis in Virus Entry and Bacterial Cell Wall Depolymerization

Abstract

Summary 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 φ29. 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 His247 is oriented to mediate nucleophile generation. This is the first model of a Zn2+-metallopeptidase and its substrate to our knowledge. 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 was maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model where 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.

PrgK, a Multidomain Peptidoglycan Hydrolase, Is Essential for Conjugative Transfer of the Pheromone-Responsive Plasmid pCF10

“…Active LytM proteins cleave the peptide cross-bridges of the PG cell wall via a reaction requiring Zn 2ϩ binding by His and Asp residues in two conserved motifs, HX 3 D and HxH (where X is any amino acid) (42,43). The degenerate LytM proteins lack one or more of the Zn 2ϩ ligands in the active site; therefore, they are catalytically inert.…”

“…S1A and S2). The N-proximal domain (residues 322 to 528) has a structure closely similar (16% identity, 100.0% confidence) to that of Staphylococcus aureus LytM (Phyre2 fold library code d1qwya), a metalloendopeptidase of the M23 family (41,42,43). The central domain (residues 532 to 720) has a structure similar (29% identity, 100.0% confidence) to that of the N terminus of bacteriophage ⌽29 tail protein gp13 (code c3csqC) and C-type goose lysozyme (20% similarity, 92.1% confidence; code d1iiza) (44).…”

A LytM Domain Dictates the Localization of Proteins to the Mother Cell-Forespore Interface during Bacterial Endospore Formation

“…These residues allow Zn 2ϩ to polarize the carbonyl carbon of the substrate for susceptibility to nucleophilic attack. A hydroxyl nucleophile generated by the His247-mediated deprotonation of water was proposed to facilitate hydrolysis, because His247 is close to the carbonyl group and in the same plane, while His291 is farther from the carbonyl group and off axis compared to His247 (S. aureus LytM numbering) (9). SpoIIQ contains only two of the three Zn 2ϩ ligands (D123 and H204 by B. subtilis SpoIIQ numbering) and lacks the proposed catalytic His ( Fig.…”

Bacillus subtilis CwlP of the SP-β Prophage Has Two Novel Peptidoglycan Hydrolase Domains, Muramidase and Cross-linkage Digesting dd-Endopeptidase

“…despite the fact that LytM and Ale1 are both Zn 2ϩ -dependent metalloendopeptidases (26,30). 29 Bacillus bacteriophage gp13 (D-alanyl-A 2 pm endopeptidase) also requires a Zn 2ϩ ion for activity (31). We conclude, therefore, that the CwlP-M23 enzyme is unique because it does not depend on Zn 2ϩ for catalytic activity.…”

“…Asterisks denote the critical amino acid residues within CwlP-M23 that were identified in this study. (31). B, in vitro cell wall hydrolytic activity.…”