Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His 6 ) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His 6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.Enzymes involved in the assembly of the peptidoglycan layer of bacterial cell walls represent important targets for antibacterial chemotherapy (15, 49). The study of this class of enzymes and the search for selective inhibitors are likely to lead to the development of new chemotherapeutic agents, which are urgently needed to combat antimicrobial drug resistance, the threat of which has recently been highlighted by the acquisition of resistance by methicillin-resistant Staphylococcus aureus to vancomycin (43).Peptidoglycan consists of a ␤-1,4-linked N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide (GlcNAc-MurNAc-pentapeptide) polymer, assembled from cytoplasmic precursors UDP-MurNAc-L-Ala-␥-D-Glu-X-D-Ala-D-Ala (UDPMurNAc-pentapeptide; X, L-Lys or meso-diaminopimelic acid [meso-DAP]) and 49

MurM is an aminoacyl ligase that adds L-serine or L-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM 159 alanylated the peptidoglycan ⑀-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM Pn16 activity supported serylation more than alanylation both in vivo and in vitro. The stem peptide is constructed in the cytoplasm appended to a UDP nucleotide (Fig.

The cost of penicillin-resistance acquisition for the Streptococcus pneumoniae strain competing with its susceptible ancestor to colonize the URT increases with the number of resistant pbp alleles acquired.

Table 1 (2, 7).The addition of the branched peptide cross-link usually occurs at the stage of lipid intermediate II (although it occurs on UDP-MurNAc-pentapeptide in Weissella viridescens (8)). Residues are added sequentially to the ⑀-amino terminus of L-lysine, in the opposite direction to that of protein synthesis (9 -13). The addition of the amino acid residues of the crosslink is catalyzed by membrane-associated ligases, which utilize aminoacyl-tRNAs as substrates (7, 13).The genetic determinants of branched wall structure in S. pneumoniae are the murM and murN genes (14, 15). MurM catalyzes the addition of L-Ala or L-Ser, whereas the addition of the second L-Ala is catalyzed by MurN (16). S. pneumoniae cell walls contain a mixture of directly linked (unbranched) and indirectly linked (branched) peptidoglycan, but the murMN genes are not essential, since direct cross-links can be formed (7,15,17). However, these enzymes do have a role in the phenotype of penicillin resistance, since inactivation of murMN leads to a loss of penicillin resistance (16,17). Clinical strains of penicillin-resistant S. pneumoniae require for the high level resistance phenotype 1) the presence of specific murMN sequences, responsible for dipeptide cross-link formation and 2) specific modified penicillin-binding protein sequences (16 -20). However, certain laboratory S. pneumoniae strains containing resistant murMN alleles do not show penicillin resistance, since they lack high affinity penicillin-binding proteins (35).The characterization of S. pneumoniae MurM ligases from a highly penicillin-resistant strain (159) and penicillin-susceptible strain (Pn16) has been recently carried out by Lloyd et al. (1), using enzymatically synthesized lipid II substrate (1, 21-23). The markedly different branching phenotype displayed by S. pneumoniae Pn16 and 159 is rationalized in vitro by the much higher specific activity of MurM 159 over MurM Pn16 with pneumococcal alanyl-tRNA Ala and the higher activity with alanyltRNA Ala than with seryl-tRNA Ser (1). In order to better understand the molecular basis of penicillin resistance caused by MurM and MurN, we wished to kinetically characterize the second ligase MurN in two clinical isolates of S. pneumoniae, one highly penicillin-resistant (159) and the other penicillin-sensitive (Pn16). In order to reconstitute

These data show that PRSP have similar TLR2-proinflammatory effects to PSSP when exposed to beta-lactam antibiotics but the antibiotic concentration relative to the MIC is critical. This has implications for treatment of pneumococcal disease when tissue concentrations of antibiotic are close to the MIC.