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-re sistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affini ty for beta-lactam antibiotics. Their transpeptidase domain carries numerou s substitutions compared with homologous sequences from beta-lactam-sensiti ve streptococci (S-PBP2x), Comparison of R-PBP2x sequences suggested that t he mutation Gln(552) --> Glu is important for resistance development. Mutan ts selected in the laboratory with cephalosporins frequently contain a muta tion Thr(550) --> Ala, The high resolution structure of a complex between S -PBP2x* and cefuroxime revealed that Gln(552) and Thr(550), which belong to strand beta 3, are in direct contact with the cephalosporin. We have studi ed the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S -PBP2x*) expressed in Escherichia coil. Mutation Q552E lowered the acylatio n efficiency for both penicillin G and cefotaxime when compared with S-PBP2 x*. We propose that the introduction of a negative charge in strand beta 3 conflicts with the negative charge of the beta-lactam. Mutation T550A lower ed the acylation efficiency of the protein for cefotaxime but not for penic illin G, The in vitro data presented here are in agreement with the distinc t resistance profiles mediated by these mutations in vivo and underline the ir role as powerful resistance determinants.