Mutations in the active site of penicillin-binding protein PBP2x from Streptococcus pneumoniae. Role in the specificity for beta-lactam antibiotics.
Penicillin-binding protein 2x (PBP2x) isolated from clinical beta-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for beta-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from beta-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 --> Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 --> Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand beta3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand beta3 conflicts with the negative charge of the beta-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. The in vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.
Mouz
N
N
Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat à l'Energie Atomique/Centre National de la Recherche Scientifique), 41, rue Jules Horowitz, 38027 Grenoble cedex, France.
Di Guilmi
A M
AM
Gordon
E
E
Hakenbeck
R
R
Dideberg
O
O
Vernet
T
T
D000215:Acylation; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002444:Cefuroxime / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010400:Penicillin G / Q000378:metabolism; D010403:Penicillin Resistance / Q000235:genetics; D046915:Penicillin-Binding Proteins; D017433:Protein Structure, Secondary; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000378:metabolism
D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; C072223:PBP 2x protein, Streptococcus; D002444:Cefuroxime; D010400:Penicillin G