Shared catalysis in virus entry and bacterial cell wall depolymerization.
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 beta-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 varphi29. 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 Zn(2)(+) metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn(2)(+) 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 Zn(2)(+) 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.
Department of Microbiology, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.
Daniel N
DN
Cohen
Yuk Y
YY
Sham
Greg D
GD
Haugstad
Ye
Y
Xiang
Michael G
MG
Rossmann
Dwight L
DL
Anderson
David L
DL
Popham
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000378:metabolism*; D001412:Bacillus subtilis / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D002473:Cell Wall / Q000378:metabolism*; D003198:Computer Simulation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053586:Virus Internalization; D015032:Zinc / Q000737:chemistry
D010455:Peptides; D010457:Peptidoglycan; D014759:Viral Envelope Proteins; D015032:Zinc