title: Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases.
abstract: The class A beta-lactamases and the transpeptidase domain of the penicillin-binding proteins (PBPs) share the same topology and conserved active-site residues. They both react with beta-lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the beta-lactams. In contrast, the deacylation of the beta-lactamases is extremely fast, resulting in a high turnover of beta-lactam hydrolysis, which confers resistance to these antibiotics. In TEM-1 beta-lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH-dependent manner, for the acidic residues. The aspartic acid-containing variant had a 110-fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.
authors.affiliation: Laboratoire d'Ingénierie des Macromolécules and Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale J.-P. Ebel, Grenoble, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Chesnel
authors.forename: André
authors.initials: A
authors.lastname: Zapun
authors.forename: Nicolas
authors.initials: N
authors.lastname: Mouz
authors.forename: Otto
authors.initials: O
authors.lastname: Dideberg
authors.forename: Thierry
authors.initials: T
authors.lastname: Vernet
mesh_terms: D000215:Acylation; D002352:Carrier Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D046915:Penicillin-Binding Proteins; D017354:Point Mutation; D013296:Streptococcus pneumoniae / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; C072223:PBP 2x protein, Streptococcus; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1