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Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.

Villet R, Fonvielle M, Busca P, Chemama M, Maillard AP, Hugonnet JE, Dubost L, Marie A, Josseaume N, Mesnage S, Mayer C, Valéry JM, Ethève-Quelquejeu M, Arthur M - Nucleic Acids Res. (2007)

Bottom Line: Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme.In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv).The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.

ABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

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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. FemXWv 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.
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Figure 2: 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. FemXWv 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.

Mentions: Synthesis of the side chain of peptidoglycan precursors was also assessed by analyzing the structure of mature peptidoglycan by mass spectrometry. Identification of 120 peptidoglycan fragments obtained by digestion of the cell wall by muramidases revealed variations in the length of stem peptides and of side chains in combination with various degree of oligomerization (Figure 1 and data not shown). Briefly, the stem of monomers consisted of a tripeptide (l-Ala1-d-iGln2-l-Lys3) or a tetrapeptide (l-Ala1-d-iGln2-l-Lys3-d-Ala4) in similar amounts indicating that the l-Lys3-d-Ala4 and d-Ala4-d-Lac5 peptide bonds were cleaved by l,d and d,d-carboxypeptidases, respectively (18). The side chains of monomers mainly consisted of the sequence l-Ala-l-Ser and l-Ala-l-Ser-l-Ala. Side chains consisting of a single l-Ala were less abundant and unsubstituted monomers were not detected. These observations indicate that FemXWv efficiently and specifically adds l-Ala to the precursors in agreement with the analysis of the cytoplasmic pool of UDP-MurNAc-peptides. The second (l-Ser) and third (l-Ala) residues of the side chain were added by unknown Fem transferases, since FemXWv adds a single residue in the in vitro coupled assay containing the accessory systems for Ala-tRNAAla and Ser-tRNASer synthesis (Figure 2 and data not shown). The second and third residues were added after the transfer of the phospho-MurNAc-peptide moiety of the precursor to the lipid carrier since a single l-Ala residue was detected in the nucleotide precursors (Figure 1). The peptidoglycan of W. viridescens contained multimeric structure ranging from dimers to hexamers. The cross-bridges contained the sequence l-Ala-l-Ser and l-Ala-l-Ser-l-Ala indicating that the d,d-transpeptidases used acceptor containing at least two residues in their side chains.Figure 2.


Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.

Villet R, Fonvielle M, Busca P, Chemama M, Maillard AP, Hugonnet JE, Dubost L, Marie A, Josseaume N, Mesnage S, Mayer C, Valéry JM, Ethève-Quelquejeu M, Arthur M - Nucleic Acids Res. (2007)

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. FemXWv 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.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175331&req=5

Figure 2: 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. FemXWv 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.
Mentions: Synthesis of the side chain of peptidoglycan precursors was also assessed by analyzing the structure of mature peptidoglycan by mass spectrometry. Identification of 120 peptidoglycan fragments obtained by digestion of the cell wall by muramidases revealed variations in the length of stem peptides and of side chains in combination with various degree of oligomerization (Figure 1 and data not shown). Briefly, the stem of monomers consisted of a tripeptide (l-Ala1-d-iGln2-l-Lys3) or a tetrapeptide (l-Ala1-d-iGln2-l-Lys3-d-Ala4) in similar amounts indicating that the l-Lys3-d-Ala4 and d-Ala4-d-Lac5 peptide bonds were cleaved by l,d and d,d-carboxypeptidases, respectively (18). The side chains of monomers mainly consisted of the sequence l-Ala-l-Ser and l-Ala-l-Ser-l-Ala. Side chains consisting of a single l-Ala were less abundant and unsubstituted monomers were not detected. These observations indicate that FemXWv efficiently and specifically adds l-Ala to the precursors in agreement with the analysis of the cytoplasmic pool of UDP-MurNAc-peptides. The second (l-Ser) and third (l-Ala) residues of the side chain were added by unknown Fem transferases, since FemXWv adds a single residue in the in vitro coupled assay containing the accessory systems for Ala-tRNAAla and Ser-tRNASer synthesis (Figure 2 and data not shown). The second and third residues were added after the transfer of the phospho-MurNAc-peptide moiety of the precursor to the lipid carrier since a single l-Ala residue was detected in the nucleotide precursors (Figure 1). The peptidoglycan of W. viridescens contained multimeric structure ranging from dimers to hexamers. The cross-bridges contained the sequence l-Ala-l-Ser and l-Ala-l-Ser-l-Ala indicating that the d,d-transpeptidases used acceptor containing at least two residues in their side chains.Figure 2.

Bottom Line: Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme.In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv).The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.

ABSTRACT
The FemX(Wv) aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l-Ala from Ala-tRNA(Ala) to UDP-MurNAc-pentadepsipeptide. FemX(Wv) is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX(Wv) is highly specific for incorporation of l-Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro-transcribed tRNAs indicated that the specificity of FemX(Wv) depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G1-C72 and G2-C71 base pairs of the acceptor stem as critical for FemX(Wv) activity in agreement with modeling of tRNA(Ala) in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA(Ala) harboring nucleotide substitutions in the G3-U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX(Wv). The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX(Wv) could be achieved by targeting the distal portion of tRNA(Ala) for the design of substrate analogues.

Show MeSH