Penicillin-binding proteins (PBPs) are the main targets for ␤-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with ␤-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level ␤-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr 338 3 Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser 389 3 Leu and Asn 514 3 His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for ␤-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.

The crystal structure of the class-B ␤-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, D-captopril, has been solved at 1.5-Å resolution. The enzyme has the typical ␣␤/␤␣ metallo-␤-lactamase fold and the characteristic two metal binding sites of members of the subclass B1, in which two Zn 2؉ ions were identified. D-Captopril, a diastereoisomer of the commercial drug, captopril, acts as an inhibitor by displacing the catalytic hydroxyl ion required for antibiotic hydrolysis and intercalating its sulfhydryl group between the two Zn 2؉ ions. Interestingly, D-captopril is located on one side of the active site cleft. The x-ray structure of the complex of the closely related enzyme, IMP-1, with a mercaptocarboxylate inhibitor, which also contains a sulfhydryl group bound to the two Zn 2؉ ions, shows the ligand to be located on the opposite side of the active site cleft. A molecule generated by fusion of these two inhibitors would cover the entire cleft, suggesting an interesting approach to the design of highly specific inhibitors.

Extensive use of ␤-lactam antibiotics has led to the selection of pathogenic streptococci resistant to ␤-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 ␤-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S 443 SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.The high-molecular-weight (high-M r ) penicillin-binding proteins (PBPs) carry out the extracellular steps of peptidoglycan synthesis. These enzymes catalyze the glycosyltransferase reaction leading to the polymerization of the glycan chains and the transpeptidation (TP) activity establishing a covalent bond between two juxtaposed peptides (3,15,32). Streptococcus pneumoniae class A high-M r PBPs (PBP1a, PBP1b, and PBP2a) are bifunctional enzymes bearing both activities, whereas class B high-M r PBPs (PBP2b and PBP2x) have so far only been associated with the TP activity (15). Due to their structural similarity to the natural substrates, the D-Ala-D-Ala stem peptides, ␤-lactam molecules inhibit the TP reaction leading to bacterial cell lysis (35).S. pneumoniae is a major human pathogen and the causative agent of ear infections in children, as well as meningitis and pneumonia. Extensive use of ␤-lactam antibiotics over the last 4 decades has led to the selection of pathogenic streptococci resistant to their action. The resistance to ␤-lactams in S. pneumoniae results from a decreased affinity of PBPs for the antibiotics. This phenotype results from genetic reshuffling. Mosaic genes encoding PBPs with lower affinity for ␤-lactams have been generated by homologous recombination events with other streptococcal species (18). The mosaicity of the pneumococcal pbp2b gene has been well established in penicillin clinical resistant (CR) isolates as resulting from recombination events between S. pneumoniae and Streptococcus mitis (8-10). DNA sequence analysis has produced evidence that, under clinical and laboratory conditions, mutants of the pbp2x and pbp2b genes are the first to be selected upon treatment with cefotaxime and pipe...