Shared catalysis in virus entry and bacterial cell wall depolymerization.

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Bacterial virus entry and cell wall lysis depend on peptidoglycan (PG) degradation.
• The enzymatic mechanism of PG hydrolysis by lysostaphin and its homologs remained unclear.
• Conserved active site residues and beta-sheet structures exist in lysostaphin, LytM, and bacteriophage gp13.

Purpose of the Study:

• To elucidate the catalytic mechanism of gene product 13 (gp13), a protein from Bacillus subtilis bacteriophage varphi29, in peptidoglycan hydrolysis.
• To investigate the role of specific active site residues (Asp195, His247) in gp13 function and Zn(2+) binding.
• To establish a shared mechanistic model for PG degradation enzymes.

Main Methods:

• Assayed gp13 activity on PG and muropeptides using high-performance liquid chromatography.
• Employed computational modeling to simulate gp13 active site interactions with a B. subtilis cross-linked peptide.
• Utilized substitution mutagenesis (Asp195 to Ala or Cys) and biophysical techniques (circular dichroism, particle-induced X-ray emission spectroscopy) to assess enzyme function and structure.

Main Results:

• gp13 was identified as a d,d-endopeptidase cleaving the peptide cross-link in PG.
• Computational modeling proposed Asp195 for scissile-bond activation and His247 for nucleophile generation in a Zn(2+) metallopeptidase model.
• Asp195 was critical for Zn(2+) binding and catalysis; the Cys mutant retained structure and Zn(2+) binding, unlike the Ala mutant.

Conclusions:

• A detailed model for the Zn(2+) metallopeptidase mechanism of gp13 was proposed, involving Asp195 and His247.
• The findings support a shared catalytic mechanism for gp13, LytM, and lysostaphin in hydrolyzing the PG peptide cross-link.
• This shared mechanism is crucial for bacterial lysis and bacteriophage propagation.