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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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Catalytic efficiency of FemXWv 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 FemXWv was rate-limiting and the tRNAs were completely acylated by the aminoacyl-tRNA synthetases, (A) FemXWv (0.5 nM), alanyl-tRNA synthetase (0.8 µM); (B) FemXWv (2 nM), seryl-tRNA synthetase (0.8 µM); (C) FemXWv (10 nM), glycyl-tRNA synthetase (0.8 µM). The FemXWv turnover numbers deduced from the kinetics were plotted as a function of the concentrations of Ala-tRNAAla (D), Ser-tRNASer (E), Gly-tRNAGly (F). The slopes (y) provide an estimate of the relative catalytic efficiencies of FemXWv for the transfer of Ala, Ser and Gly at non-saturating concentrations of aminoacyl-tRNAs (turnover per min and per µM of tRNAs).
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Figure 3: Catalytic efficiency of FemXWv 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 FemXWv was rate-limiting and the tRNAs were completely acylated by the aminoacyl-tRNA synthetases, (A) FemXWv (0.5 nM), alanyl-tRNA synthetase (0.8 µM); (B) FemXWv (2 nM), seryl-tRNA synthetase (0.8 µM); (C) FemXWv (10 nM), glycyl-tRNA synthetase (0.8 µM). The FemXWv turnover numbers deduced from the kinetics were plotted as a function of the concentrations of Ala-tRNAAla (D), Ser-tRNASer (E), Gly-tRNAGly (F). The slopes (y) provide an estimate of the relative catalytic efficiencies of FemXWv for the transfer of Ala, Ser and Gly at non-saturating concentrations of aminoacyl-tRNAs (turnover per min and per µM of tRNAs).

Mentions: In order to compare the catalytic efficiency of FemXWv with different aminoacyl-tRNAs, kinetics of synthesis of UDP-MurNAc-hexapeptides containing Ala (Figure 3A), Ser (Figure 3B), and Gly (Figure 3C) were performed at different aminoacyl-tRNA concentrations. The turnover number of FemXWv was deduced from the individual kinetics and plotted against the aminoacyl-tRNA concentrations (Figure 3D–F). The linear relationship indicates that the reaction conditions meet the first order conditions with respect to aminoacyl-tRNAs in agreement with the Km value of 1.7 µM previously reported for Ala-tRNAAla (13). Ser-tRNASer and Gly-tRNAGly were used 17- and 38-fold less efficiently than Ala-tRNAAla by FemXWv (36 and 16 versus 605 min−1µM−1, respectively; Table 1).Figure 3.


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)

Catalytic efficiency of FemXWv 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 FemXWv was rate-limiting and the tRNAs were completely acylated by the aminoacyl-tRNA synthetases, (A) FemXWv (0.5 nM), alanyl-tRNA synthetase (0.8 µM); (B) FemXWv (2 nM), seryl-tRNA synthetase (0.8 µM); (C) FemXWv (10 nM), glycyl-tRNA synthetase (0.8 µM). The FemXWv turnover numbers deduced from the kinetics were plotted as a function of the concentrations of Ala-tRNAAla (D), Ser-tRNASer (E), Gly-tRNAGly (F). The slopes (y) provide an estimate of the relative catalytic efficiencies of FemXWv for the transfer of Ala, Ser and Gly at non-saturating concentrations of aminoacyl-tRNAs (turnover per min and per µM of tRNAs).
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Figure 3: Catalytic efficiency of FemXWv 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 FemXWv was rate-limiting and the tRNAs were completely acylated by the aminoacyl-tRNA synthetases, (A) FemXWv (0.5 nM), alanyl-tRNA synthetase (0.8 µM); (B) FemXWv (2 nM), seryl-tRNA synthetase (0.8 µM); (C) FemXWv (10 nM), glycyl-tRNA synthetase (0.8 µM). The FemXWv turnover numbers deduced from the kinetics were plotted as a function of the concentrations of Ala-tRNAAla (D), Ser-tRNASer (E), Gly-tRNAGly (F). The slopes (y) provide an estimate of the relative catalytic efficiencies of FemXWv for the transfer of Ala, Ser and Gly at non-saturating concentrations of aminoacyl-tRNAs (turnover per min and per µM of tRNAs).
Mentions: In order to compare the catalytic efficiency of FemXWv with different aminoacyl-tRNAs, kinetics of synthesis of UDP-MurNAc-hexapeptides containing Ala (Figure 3A), Ser (Figure 3B), and Gly (Figure 3C) were performed at different aminoacyl-tRNA concentrations. The turnover number of FemXWv was deduced from the individual kinetics and plotted against the aminoacyl-tRNA concentrations (Figure 3D–F). The linear relationship indicates that the reaction conditions meet the first order conditions with respect to aminoacyl-tRNAs in agreement with the Km value of 1.7 µM previously reported for Ala-tRNAAla (13). Ser-tRNASer and Gly-tRNAGly were used 17- and 38-fold less efficiently than Ala-tRNAAla by FemXWv (36 and 16 versus 605 min−1µM−1, respectively; Table 1).Figure 3.

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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