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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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Recognition of tRNAs by alanyl-tRNA synthetases and FemXWv. (A) Sequence of the tRNAAla acceptor stem. The bases essential for formation of UDP-MurNAc-hexapeptide in the coupled AlaRS-FemXWv assay are boxed. Among these four bases, only C72 and C71 are essential for FemXWv activity as shown by comparing the impact of substitutions in the coupled assay and the direct determination of FemXWv activity using semi-synthetic substrates (right panel). (B) Model of FemXWv-tRNAAla interaction showing domains I (blue) and II (pink) of FemXWv and the tRNAAla (red) and UDP-MurNAc-pentapeptide (green) substrates.
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Figure 8: Recognition of tRNAs by alanyl-tRNA synthetases and FemXWv. (A) Sequence of the tRNAAla acceptor stem. The bases essential for formation of UDP-MurNAc-hexapeptide in the coupled AlaRS-FemXWv assay are boxed. Among these four bases, only C72 and C71 are essential for FemXWv activity as shown by comparing the impact of substitutions in the coupled assay and the direct determination of FemXWv activity using semi-synthetic substrates (right panel). (B) Model of FemXWv-tRNAAla interaction showing domains I (blue) and II (pink) of FemXWv and the tRNAAla (red) and UDP-MurNAc-pentapeptide (green) substrates.

Mentions: To identify the tRNA elements essential for FemXWv activity, deletions were introduced into tRNAAla revealing that the acceptor stem is sufficient for the alanine transferase activity of FemXWv (Figure 4). Site-directed mutagenesis identified four bases (C72, C71 and G3–U70) essential for UDP-MurNAc-hexapeptide synthesis in the coupled AlaRS-FemXWv assay (Figure 5). Introduction of double nucleotide substitutions showed that C72 and C71 were both essential since destabilization of the acceptor stem was not the primary impact of nucleotide substitutions at these two positions (Figure 6). Site-directed mutagenesis (Figures 5 and 6) combined to semi-synthesis (Figure 7) showed that the identity determinants of the E. faecalis AlaRS and FemXWv are overlapping but distinct (Figure 8A). In particular, the G3–U70 base pair of tRNAAla, which is the main identity determinant of AlaRS from bacteria, archaea and eukarya (31,32), occasionally found at positions 2–71 (33,34), was non-essential to the alanyl transferase activity of FemXWv since nucleotide substitutions at positions 3 and 70 were both tolerated by the enzyme (Figure 8A). Modeling of tRNAAla in the FemXWv active site (Figure 8B) indicated that the first two base pairs (G1–C72 and G2–C71) of the acceptor stem and the ACCA end are likely to be in close contact with the protein. The lack of direct interaction between the 3–70 base pair and FemXWv is in agreement with the non-essential nature of G3 and U70 for the alanyl transferase activity of this enzyme. Substitutions that restored Watson–Crick base pairing at position 3–70 did not abolish FemXWv activity. Thus, indirect recognition of the wobble base pair through a structural perturbation in the acceptor stem (34,35) appears also to be excluded for FemXWv.Figure 8.


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)

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

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Figure 8: Recognition of tRNAs by alanyl-tRNA synthetases and FemXWv. (A) Sequence of the tRNAAla acceptor stem. The bases essential for formation of UDP-MurNAc-hexapeptide in the coupled AlaRS-FemXWv assay are boxed. Among these four bases, only C72 and C71 are essential for FemXWv activity as shown by comparing the impact of substitutions in the coupled assay and the direct determination of FemXWv activity using semi-synthetic substrates (right panel). (B) Model of FemXWv-tRNAAla interaction showing domains I (blue) and II (pink) of FemXWv and the tRNAAla (red) and UDP-MurNAc-pentapeptide (green) substrates.
Mentions: To identify the tRNA elements essential for FemXWv activity, deletions were introduced into tRNAAla revealing that the acceptor stem is sufficient for the alanine transferase activity of FemXWv (Figure 4). Site-directed mutagenesis identified four bases (C72, C71 and G3–U70) essential for UDP-MurNAc-hexapeptide synthesis in the coupled AlaRS-FemXWv assay (Figure 5). Introduction of double nucleotide substitutions showed that C72 and C71 were both essential since destabilization of the acceptor stem was not the primary impact of nucleotide substitutions at these two positions (Figure 6). Site-directed mutagenesis (Figures 5 and 6) combined to semi-synthesis (Figure 7) showed that the identity determinants of the E. faecalis AlaRS and FemXWv are overlapping but distinct (Figure 8A). In particular, the G3–U70 base pair of tRNAAla, which is the main identity determinant of AlaRS from bacteria, archaea and eukarya (31,32), occasionally found at positions 2–71 (33,34), was non-essential to the alanyl transferase activity of FemXWv since nucleotide substitutions at positions 3 and 70 were both tolerated by the enzyme (Figure 8A). Modeling of tRNAAla in the FemXWv active site (Figure 8B) indicated that the first two base pairs (G1–C72 and G2–C71) of the acceptor stem and the ACCA end are likely to be in close contact with the protein. The lack of direct interaction between the 3–70 base pair and FemXWv is in agreement with the non-essential nature of G3 and U70 for the alanyl transferase activity of this enzyme. Substitutions that restored Watson–Crick base pairing at position 3–70 did not abolish FemXWv activity. Thus, indirect recognition of the wobble base pair through a structural perturbation in the acceptor stem (34,35) appears also to be excluded for FemXWv.Figure 8.

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