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In vitro Reconstitution of Peptidoglycan Assembly from the Gram-Positive Pathogen Streptococcus pneumoniae
journal contribution
posted on 2013-12-20, 00:00 authored by André Zapun, Jules Philippe, Katherine A. Abrahams, Luca Signor, David I. Roper, Eefjan Breukink, Thierry VernetUnderstanding
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.