Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis

Abstract

Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemX(Wv). Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemX(Wv) with the acceptor arm of tRNA(Gly) and with L-Ser or larger residues quantitatively accounts for the preferential transfer of L-Ala observed with complete aminoacyl-tRNAs. The main FemX(Wv) identity determinant was identified as the penultimate base pair (G(2)-C(71)) of the acceptor arm instead of G(3)*U(70) for the alanyl-tRNA synthetase. FemX(Wv) tolerated a configuration inversion of the Calpha of L-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemX(Wv) is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNA(Gly) from the active site.

Figure 1.

Peptidoglycan synthesis pathway in W. viridescens. The FemX_Wv transferase adds the first residue of the l-Ala-l-Ser-l-Ala side chain onto the nucleotide precursor UDP-MurNAc-pentadepsipeptide. The Ala-tRNA^Ala substrate of FemX_Wv is produced by the alanyl-tRNA-synthetase (AlaRS). Additional unknown Fem transferases add the second (l-Ser) and third (l-Ala) residues of the side chain onto the precursors linked to the undecaprenyl lipid carrier (30). Black box, undecaprenyl; d-Lac, d-lactate; lipid II, undecaprenyl-diphospho-MurNAc(pentadepsipeptide)-GlcNAc. d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family catalyse formation of a peptide bound between the carbonyl of d-Ala⁴ of an acyl donor stem and the amine at the side chain extremity of an acceptor stem.

Figure 2.

Semi-synthesis of Ala-tRNA^Ala analogues. (A) Organic synthesis of dinucleotides acylated by Gly, l-Ala, d-Ala and Abu protected with a pentenoyl group. pdCpA, (5′)phospho(2′)deoxycytidine-(5′)phosphoadenine. (B) Organic synthesis of pdCpA acylated by l-Ser protected with a pentenoyl group. (C) Ligation of acylated dinucleotides to RNA helix^Ala. The product of the reaction catalysed by the T4 RNA ligase was deprotected with iodine.

Figure 3.

Specificity of FemX_Wv for the aminoacyl residue. (A) The relative efficiency of FemX_Wv was estimated for the transfer of l-Ala, l-Ser and Gly from acylated RNA helix^Ala to UDP-MurNAc-[¹⁴C]pentapeptide. (B) The radiolabelled substrate and product of the reaction catalysed by FemX_Wv were determined by rp-HPLC coupled to a radioflow detector. The superimposed chromatograms provide examples of the separation of the UDP-MurNAc-[¹⁴C]hexapeptide product containing l-Ala (a), l-Ser (b) or Gly (c) from the UDP-MurNAc-[¹⁴C]pentapeptide substrate (d) common to the three reactions. CTS, counts per s. (C) The initial concentration of acylated RNA helix^Ala in each reaction was estimated following complete transfer of the residue at high concentrations of FemX_Wv (after completion of the aminoacyl transfer reaction, the concentration of UDP-MurNAc-[¹⁴C]hexapeptide was equal to the initial concentration of the Ala-helix^Ala). (D) At lower concentrations of FemX_Wv, the extent of the transfer was proportional to the concentration of FemX_Wv. (E) The slopes were used to estimate the relative efficiency of the enzyme for the different acylated helices.

Figure 4.

Specificity of FemX_Wv for the sequence of the acceptor arm of tRNA^Ala, tRNA^Ser and tRNA^Gly. (A) RNA sequence of the helices. Nucleotide substitutions introduced into helix^Ala in order to obtain helix^Ser and helix^Gly are boxed. The sequence of the distal portion of the helix^Ser and helix^Gly (base pairs 1–72, 2–71, 3–70 and unpaired bases 73–76) were design according to the sequence of the acceptor arm of tRNA^Ser and tRNA^Gly. (B and C) The relative efficiency of FemX_Wv was estimated for the transfer of l-Ala from the RNA helices to UDP-MurNAc-[¹⁴C]pentapeptide.

Figure 5.

Inhibition of FemX_Wv by substrate analogues. (A) Structure of the Ala-helix^Ala and analogues containing an oxadiazole moiety linked to helix^Ala, helix^Ser and helix^Gly. (B) IC₅₀ values were determined in the AlaRS-FemX_Wv coupled assay.

Figure 6.

Specificity of FemX_Wv for the discriminator base and the first three base pairs of the acceptor arm. (A) Nucleotide substitutions were introduced into the distal portion of the RNA helix^Ala (boxed). (B and C) The relative efficiency of FemX_Wv was estimated for the transfer of l-Ala from the acylated helices to UDP-MurNAc-[¹⁴C]pentapeptide. Data are presented in two graphs according to the range of FemX_Wv concentrations required to estimate enzyme activity.

Figure 7.

Stereospecificity of FemX_Wv. The relative efficiency of FemX_Wv was estimated for the transfer of l-Ala, d-Ala and 2-amino-isobutyric acid (Abu) from helix^Ala to UDP-MurNAc-[¹⁴C]pentapeptide. A modified acetonitrile gradient (0–20%) followed by a step elution at 100% was used to confirm the absence of the transfer of Abu since the corresponding UDP-MurNAc-hexapeptide is expected to be hydrophobic and might have been retained in the column under the classical conditions.

Figure 8.

Relative contribution of the aminoacyl residue and of the nucleotide sequence to the specificity of FemX_Wv. Previous comparisons of full tRNAs obtained by in vitro transcription indicated that Ser-tRNA^Ser and Gly-tRNA^Gly are used 17- and 38-fold less efficiently than Ala-tRNA^Ala by FemX_Wv (6.0% and 2.6%, respectively) (30). In this study, the main identity determinants of FemX_Wv were identified based on independently testing the impact of substitutions of the aminoacyl residue and of nucleotides on FemX_Wv activity. Substitutions were chosen according to the differences found in the distal portion of the acceptor arm of tRNA^Ala, tRNA^Ser and tRNA^Gly. The discrimination between l-Ala and l-Ser is the main factor that prevents incorporation of l-Ser by FemX_Wv whereas the C²-G⁷¹ base pair in the acceptor arm of tRNA^Gly is the major anti-determinant that prevents incorporation of Gly.