title: The membrane anchor of penicillin-binding protein PBP2a from Streptococcus pneumoniae influences peptidoglycan chain length.
abstract: The pneumococcus is an important Gram-positive pathogen, which shows increasing resistance to antibiotics, including β-lactams that target peptidoglycan assembly. Understanding cell-wall synthesis, at the molecular and cellular level, is essential for the prospect of combating drug resistance. As a first step towards reconstituting pneumococcal cell-wall assembly in vitro, we present the characterization of the glycosyltransferase activity of penicillin-binding protein (PBP)2a from Streptococcus pneumoniae. Recombinant full-length membrane-anchored PBP2a was purified by ion-exchange chromatography. The glycosyltransferase activity of this enzyme was found to differ from that of a truncated periplasmic form. The full-length protein with its cytoplasmic and transmembrane segment synthesizes longer glycan chains than the shorter form. The transpeptidase active site was functional, as shown by its reactivity towards bocillin and the catalysis of the hydrolysis of a thiol-ester substrate analogue. However, PBP2a did not cross-link the peptide stems of glycan chains in vitro. The absence of transpeptidase activity indicates that an essential component is missing from the in vitro system.
authors.affiliation: CEA, Institut de Biologie Structurale, Grenoble, France.
authors.forename: Nordine
authors.initials: N
authors.lastname: Helassa
authors.forename: Waldemar
authors.initials: W
authors.lastname: Vollmer
authors.forename: Eefjan
authors.initials: E
authors.lastname: Breukink
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
authors.forename: André
authors.initials: A
authors.lastname: Zapun
mesh_terms: D020134:Catalytic Domain; D002473:Cell Wall / Q000201:enzymology* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli; D006023:Glycoproteins / Q000096:biosynthesis; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000201:enzymology*
chemical_list: D006023:Glycoproteins; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; C478699:penicillin-binding protein 2a, Streptococcus; D010458:Peptidyl Transferases; D010453:Peptide Synthases