In vitro Reconstitution of Peptidoglycan Assembly from the Gram-Positive Pathogen Streptococcus pneumoniae

• DOI: 10.1021/cb400575t
• Publication Date: 2013-09-18T11:46:50Z
• Authors: André Zapun; Jules Philippe; Katherine A. Abra...; Luca Signor; David I. Roper; Eefjan Breukink; Thierry Vernet
• Keywords: 0301 basic medicine; 570; MESH: Peptidyl Transferases; Glutamine; Glutamic Acid; Peptidoglycan; beta-Lactams; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry; beta-Lactam Resistance; MESH: Homologous Recombination; 03 medical and health sciences; MESH: Cell Wall; MESH: beta-Lactams; Cell Wall; MESH: Anti-Bacterial Agents; 616; Penicillin-Binding Proteins; Homologous Recombination; MESH: Glutamine; MESH: Penicillin-Binding Proteins; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]; Molecular Biology/Structural Biology [q-bio.BM]; MESH: Peptidoglycan; MESH: Glutamic Acid; MESH: beta-Lactam Resistance; Uridine Diphosphate N-Acetylmuramic Acid; Anti-Bacterial Agents; 3. Good health; Streptococcus pneumoniae; Peptidyl Transferases; MESH: Uridine Diphosphate N-Acetylmuramic Acid; MESH: Genetic Engineering; MESH: Peptidoglycan Glycosyltransferase; Peptidoglycan Glycosyltransferase; Genetic Engineering; MESH: Streptococcus pneumoniae

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.