Kinetic Characterization of Lipid II-Ala:Alanyl-tRNA Ligase (MurN) from Streptococcus pneumoniae using Semisynthetic Aminoacyl-lipid II Substrates

Abstract

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

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

“…One feature contributing to this variation is the presence of both linear and branched stem peptides, the latter of which are formed by products of the murMN operon ( Fig 2A ) [ 17 ]. MurM and MurN act sequentially to catalyze the tRNA-dependent addition of a dipeptide onto the lysine residue of a PG precursor molecule; MurM acts first to add either a serine or alanine residue, which provides the substrate for the MurN-dependent addition of an alanine [ 17 – 19 ]. Deletion of murMN has no effect on growth in vitro or the apparent amount of crosslinking within PG, yet manipulation of the murMN operon causes drastic changes in the composition of stem peptides and the type of crosslinks that connect them [ 17 , 24 ].…”

“…Formation of branched stem peptides occurs through addition of a dipeptide branch on the third position lysine residue of a nascent PG precursor molecule during the membrane-associated steps of PG biosynthesis. This activity is catalyzed by two gene products encoded within the murMN operon [ 17 – 19 ]. Crosslink formation between the dipeptide branch and an adjacent stem peptide results in formation of a crossbridge, thus incorporating branched stem peptides into the existing PG network.…”

Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant

“…8, Table 3 and Table S2). First, branched muropeptides were abundant, and these contain one alanine attached to lysine instead of an Ala-Ala or Ser-Ala dipeptide found in S. pneumoniae (Garcia-Bustos et al, 1987;Garcia-Bustos and Tomasz, 1990b;De Pascale et al, 2008;Bui et al, 2012), in agreement with the lack of murN. Although the MS analysis cannot distinguish whether the Ala resides at the epsilon amino group of Lys or at position 4 of the stem peptide, the elution time of the peaks in the HPLC analysis is consistent with branched muropeptides rather than with linear ones .…”

Minimal Peptidoglycan (PG) Turnover in Wild-Type and PG Hydrolase and Cell Division Mutants of Streptococcus pneumoniae D39 Growing Planktonically and in Host-Relevant Biofilms

“…The MurMN enzymes catalyze cross-bridge formation in D39 and other pneumococcal strains (60)(61)(62)(63). We showed previously that laboratory strain R6 contains substantially more cross-bridges than its D39 progenitor strain (40).…”