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J Biol Chem, Vol. 274, Issue 27, 19175-19180, July 2, 1999

Mutations in the Active Site of Penicillin-binding Protein PBP2x from Streptococcus pneumoniae
ROLE IN THE SPECIFICITY FOR -LACTAM ANTIBIOTICS

Nicolas Mouz^§, Anne Marie Di Guilmi^§, Elspeth Gordon, Regine Hakenbeck^¶, Otto Dideberg, and Thierry Vernet^§

From the ^§ Laboratoire d'Ingénierie des Macromolécules and the  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, and the ^¶ Universität Kaiserslautern, Department of Microbiology, Paul Ehrlich Strasse, D-67663 Kaiserslautern, Germany

Penicillin-binding protein 2x (PBP2x) isolated from clinical -lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for -lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from -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 3, 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 3 conflicts with the negative charge of the -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.

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