Main steps of peptidoglycan synthesis in W. viridescens. The subunit consists of β-1-4-linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) substituted by a depsipeptide which is linked to the d-lactoyl group of MurNAc by an amide bond. The assembly of the subunit starts in the cytoplasm by the synthesis of UDP-MurNAc, the first precursor dedicated to peptidoglycan synthesis (37). In the following steps, the Mur ligases sequentially add l-Ala, d-Glu, l-Lys, attached to the γ carboxyl of d-Glu (d-iGlu) and the depsipeptide d-Ala-d-Lac to form the stem pentadepsipeptide l-Ala¹-d-iGlu²-l-Lys³-d-Ala⁴-d-Lac⁵. The FemX_Wv aminoacyl transferase adds the first residue of the side chain onto this nucleotide precursor. Synthesis of the subunit proceeds by the transfer of the phospho-MurNAc-pentadepsipeptide moiety of UDP-MurNAc-pentadepsipeptide to the C₅₅ lipid carrier undecaprenyl phosphate to form lipid intermediate I (undecaprenyl-PP-MurNAc-pentadepsipeptide or lipid I). The addition of GlcNAc to lipid I leads to lipid intermediate II [undecaprenyl-PP-MurNAc-(pentadepsipeptide)GlcNAc] or lipid II. The second and third residues of the l-Ala-l-Ser and l-Ala-l-Ser-l-Ala side chains are added to the lipid intermediates by unknown Fem transferases. The α carboxyl of d-iGlu² is amidated in mature peptidoglycan (d-iGln²). The insets indicate the relative abundance of precursors and muropeptides, which was determined by the absorbance at 260 and 195 nm, respectively.

Catalytic efficiency of FemX_Wv with different tRNAs obtained by in vitro transcription. Kinetics of transfer of Ala (A), Ser (B) and Gly (C) from aminoacyl-tRNAs to UDP-MurNAc-pentapeptide was determined with four concentrations of tRNAs (filled square, 0 µM; filled circle, 0.025 µM; open circle, 0.05 µM; open square, 0.075 µM; open circle, 0.1 µM). The concentrations of the two enzymes in the coupled assay were adjusted to obtain initial velocities in conditions where the transferase activity of FemX_Wv was rate-limiting and the tRNAs were completely acylated by the aminoacyl-tRNA synthetases, (A) FemX_Wv (0.5 nM), alanyl-tRNA synthetase (0.8 µM); (B) FemX_Wv (2 nM), seryl-tRNA synthetase (0.8 µM); (C) FemX_Wv (10 nM), glycyl-tRNA synthetase (0.8 µM). The FemX_Wv turnover numbers deduced from the kinetics were plotted as a function of the concentrations of Ala-tRNA^Ala (D), Ser-tRNA^Ser (E), Gly-tRNA^Gly (F). The slopes (y) provide an estimate of the relative catalytic efficiencies of FemX_Wv for the transfer of Ala, Ser and Gly at non-saturating concentrations of aminoacyl-tRNAs (turnover per min and per µM of tRNAs).

Residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemX_Wv coupled assay with derivatives of tRNA^Ala (76-nt) harboring single base substitutions in the acceptor stem. The standard assay was performed with AlaRS (800 nM), FemX_Wv (500 nM), l-[¹⁴C]Ala (50 µM) and the different tRNAs (ca. 5.0 µM). The reaction was incubated for 2 h at 30°C. Under these conditions, total l-[¹⁴C]Ala (0.5 nmol) was incorporated into UDP-MurNAc-hexapeptide when RNA with the wild-type sequence was used (100%). ND, not detected.

Semi-synthesis of Ala-tRNA^Ala analogues. (A) The protected dinucleotide pdCpA-Ala-pentenoyl was ligated to a 74-nt in vitro transcript by the T4 RNA ligase. The product of the reaction was deprotected leading to an acylated-RNA molecule identical to Ala-tRNA^Ala except for the presence of a 3′deoxycytidine instead of a cytidine at position 75. Nucleotide substitutions were introduced in the 74-nt in vitro transcript using the PCR approach described above except that one of the two mutagenic primers lacked the two 5′ bases specifying bases 75 and 76 of the transcript. (B) Schematic representation of the main steps for synthesis of the dinucleotide pdCpA-Ala-pentenoyl. (C) Analysis of the T4 ligation products by denaturing polyacrylamide electrophoresis. Lane 1, 76-nt transcript with the wild-type tRNA^Ala sequence; lane 2, 74-nt in vitro transcript; lane 3, ligation of the 74-nt transcript with pdCpA-Ala-pentenoyl.

Coupled assay for UDP-MurNAc-hexapeptide synthesis. The auxiliary system used to generate aminoacyl-tRNA (AA-tRNA) involves acylation of tRNA with a radiolabeled amino acid by aminoacyl-tRNA synthetases (AA-RS). Purified alanyl-tRNA synthetase (AlaRS), seryl-tRNA synthetase (SerRS) and glycyl-tRNA synthetase (GlyRS) were used in this study. FemX_Wv transfers the amino acids from the aminoacyl-tRNAs to the ɛ -amino group of l-Lys at the third position of UDP-MurNAc-pentapeptide to form the corresponding hexapeptide which was determined by liquid scintillation.

Functional RNA molecules in the synthesis of UDP-MurNAc-hexapeptide by AlaRS and FemX_Wv in the coupled assay. The 76-nt RNA corresponds to the only tRNA^Ala of E. faecalis. The minimal RNA substrate in the AlaRS-FemX_Wv coupled assay was a duplex (7 + 11 nt) mimicking the acceptor stem of the tRNA^Ala.

Histogram showing residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemX_Wv coupled assay with derivatives of tRNA^Ala (76-nt) harboring all possible combinations in base pairs G¹–C⁷² (A), G²–C⁷¹ (B), G³–U⁷⁰ (C) and G⁴–C⁶⁹ (D). The standard assay was performed with AlaRS (800 nM), FemX_Wv (500 nM), l-[¹⁴C]Ala (50 µM) and the different tRNAs (ca. 5 µM). The reaction was incubated for 2 h at 30°C. Under these conditions, total l-[¹⁴C]Ala (0.5 nmol) was incorporated into UDP-MurNAc-hexapeptide when RNA with the wild type sequence was used (100%, indicated by a star).

Recognition of tRNAs by alanyl-tRNA synthetases and FemX_Wv. (A) Sequence of the tRNA^Ala acceptor stem. The bases essential for formation of UDP-MurNAc-hexapeptide in the coupled AlaRS-FemX_Wv assay are boxed. Among these four bases, only C⁷² and C⁷¹ are essential for FemX_Wv activity as shown by comparing the impact of substitutions in the coupled assay and the direct determination of FemX_Wv activity using semi-synthetic substrates (right panel). (B) Model of FemX_Wv-tRNA^Ala interaction showing domains I (blue) and II (pink) of FemX_Wv and the tRNA^Ala (red) and UDP-MurNAc-pentapeptide (green) substrates.