In vitro reconstitution of peptidoglycan assembly from the Gram-positive pathogen Streptococcus pneumoniae

ACS Chem Biol. 2013 Dec 20;8(12):2688-96. doi: 10.1021/cb400575t. Epub 2013 Sep 30.


Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with β-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by β-lactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient cross-linking of the peptidoglycan.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Cell Wall / chemistry
  • Cell Wall / drug effects
  • Cell Wall / enzymology*
  • Cell Wall / genetics
  • Genetic Engineering
  • Glutamic Acid / metabolism
  • Glutamine / metabolism
  • Homologous Recombination
  • Penicillin-Binding Proteins / chemistry
  • Penicillin-Binding Proteins / genetics
  • Penicillin-Binding Proteins / metabolism*
  • Peptidoglycan / biosynthesis*
  • Peptidoglycan / chemistry
  • Peptidoglycan / genetics
  • Peptidoglycan Glycosyltransferase / chemistry
  • Peptidoglycan Glycosyltransferase / genetics
  • Peptidoglycan Glycosyltransferase / metabolism*
  • Peptidyl Transferases / chemistry
  • Peptidyl Transferases / genetics
  • Peptidyl Transferases / metabolism*
  • Streptococcus pneumoniae / chemistry
  • Streptococcus pneumoniae / drug effects
  • Streptococcus pneumoniae / enzymology*
  • Streptococcus pneumoniae / genetics
  • Uridine Diphosphate N-Acetylmuramic Acid / analogs & derivatives
  • Uridine Diphosphate N-Acetylmuramic Acid / chemistry
  • Uridine Diphosphate N-Acetylmuramic Acid / metabolism
  • beta-Lactam Resistance / drug effects
  • beta-Lactam Resistance / genetics
  • beta-Lactams / pharmacology


  • Anti-Bacterial Agents
  • Penicillin-Binding Proteins
  • Peptidoglycan
  • Uridine Diphosphate N-Acetylmuramic Acid
  • beta-Lactams
  • muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol
  • Glutamine
  • Glutamic Acid
  • Peptidyl Transferases
  • Peptidoglycan Glycosyltransferase