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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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Histogram showing residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (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), FemXWv (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).
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Figure 6: Histogram showing residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (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), FemXWv (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).

Mentions: All 15 possible combinations of nucleotides were introduced in base pairs G1–C72, G2–C71, G3–U70 and G4–C69 using pairs of mutagenic primers and residual activity was determined in the coupled AlaRS-FemXWv (Figure 6). For the G1–C72 base pair, C at position 72, rather than a Watson–Crick base pair, was essential for enzyme activity since tRNAs containing C1–G72, A1–U72, U1–A72 did not support UDP-MurNAc-hexapeptide synthesis. In the following base pair, residual activity was only detected for two of the 15 combinations, A2–C71 and C2–C71, revealing again that destabilization of the acceptor stem is not the main factor that determines loss of activity. At position G3–U70, none of the 15 combinations were tolerated. Finally, synthesis of UDP-MurNAc-hexapeptide was obtained with the set of 16 tRNAs containing all base combinations at positions 4 and 69. These results confirmed that four specific bases, C72, C71 and the G3–U70 pair, are essential for UDP-MurNAc-hexapeptide synthesis. None of the double substitutions that restored Watson–Crick base pairs compensated for substitution at C72 and C71. Thus, modification of the sequence rather than destabilization of the acceptor stem was the primary impact of nucleotide substitutions at these two positions.Figure 6.


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)

Histogram showing residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (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), FemXWv (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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Histogram showing residual (%) UDP-MurNAc-hexapeptide synthesis in the AlaRS-FemXWv coupled assay with derivatives of tRNAAla (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), FemXWv (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).
Mentions: All 15 possible combinations of nucleotides were introduced in base pairs G1–C72, G2–C71, G3–U70 and G4–C69 using pairs of mutagenic primers and residual activity was determined in the coupled AlaRS-FemXWv (Figure 6). For the G1–C72 base pair, C at position 72, rather than a Watson–Crick base pair, was essential for enzyme activity since tRNAs containing C1–G72, A1–U72, U1–A72 did not support UDP-MurNAc-hexapeptide synthesis. In the following base pair, residual activity was only detected for two of the 15 combinations, A2–C71 and C2–C71, revealing again that destabilization of the acceptor stem is not the main factor that determines loss of activity. At position G3–U70, none of the 15 combinations were tolerated. Finally, synthesis of UDP-MurNAc-hexapeptide was obtained with the set of 16 tRNAs containing all base combinations at positions 4 and 69. These results confirmed that four specific bases, C72, C71 and the G3–U70 pair, are essential for UDP-MurNAc-hexapeptide synthesis. None of the double substitutions that restored Watson–Crick base pairs compensated for substitution at C72 and C71. Thus, modification of the sequence rather than destabilization of the acceptor stem was the primary impact of nucleotide substitutions at these two positions.Figure 6.

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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