Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan

Abstract

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.

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

“…All proteins were overexpressed in E. coli strain BL21 (DE3) by the addition of a final concentration of 1 mM isopropyl-␤-D-1-thigalactopyranoside at an A 600 of 04 -0.6 followed by a reduction in growth temperature from 37°C to 28°C for 3-5 h. Proteins were purified on BD TALON cobalt resin using equilibration/wash buffer (50 mM sodium phosphate, pH 7.2, 500 mM sodium chloride, and 20% glycerol) containing 250 mM imidazole. MurM was solubilized prior to purification as described (21,38). S. pneumoniae tRNA Ala and tRNA Phe (wild type and U4G) and the E. coli wild type equivalents were produced by in vitro T7 RNA polymerase runoff transcription as described (22).…”

“…MurM catalyzes the transfer of either alanine or serine to the stem peptide lysine of Lipid II and, in combination with MurN, generates the substrate for indirect cross-linking of peptidoglycan. Until recently, the only aminoacyl-tRNA substrates recognized by MurM were thought to be Ser-tRNA Ser , provided by seryl-tRNA synthetase, and Ala-tRNA Ala , provided by AlaRS (38). However, MurM is also able to efficiently transfer serine to Lipid II from misaminoacylated Ser-tRNA Ala , which is also produced by pneumococcal AlaRS (21).…”

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…Lipid II Cleavage Assay-Lipid II hydrolysis assays were performed using a modified version of the assay described by Barreteau et al (35), with non-radiolabeled lipid II substrate obtained from the UK Bacterial Cell Wall Biosynthesis Network (36,37). Assays were carried out in 100 mM Tris, 150 mM NaCl, 0.2% (w/v) n-dodecyl-D-maltoside with 2.6 nmol of lipid II and 0.33 nmol of wild-type or mutant syringacin M. To test the metal dependence of syringacin M, assays were performed in the presence of MgCl 2 , CaCl 2 , MnCl 2 , CoCl 2 , ZnCl 2 , or NiSO 4 at 20 mM or with 1 mM EGTA.…”

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

“…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)(22)(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).…”