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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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Functional RNA molecules in the synthesis of UDP-MurNAc-hexapeptide by AlaRS and FemXWv in the coupled assay. The 76-nt RNA corresponds to the only tRNAAla of E. faecalis. The minimal RNA substrate in the AlaRS-FemXWv coupled assay was a duplex (7 + 11 nt) mimicking the acceptor stem of the tRNAAla.
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Figure 4: Functional RNA molecules in the synthesis of UDP-MurNAc-hexapeptide by AlaRS and FemXWv in the coupled assay. The 76-nt RNA corresponds to the only tRNAAla of E. faecalis. The minimal RNA substrate in the AlaRS-FemXWv coupled assay was a duplex (7 + 11 nt) mimicking the acceptor stem of the tRNAAla.

Mentions: Various derivatives of the E. faecalis tRNAAla (Figure 4) were tested as substrates of AlaRS and FemXWv in the coupled assay (Figure 2) for UDP-MurNAc-hexapeptide synthesis. Deletion of the anticodon and pseudouridine arms of tRNAAla resulted in two minihelices, with or without bulged U8, which were both functional in the coupled assay. Formation of UDP-MurNAc-hexapeptide was also obtained with a 24-nt microhelix consisting of the acceptor stem of tRNAAla and a loop of 6 nt. Finally, an RNA duplex consisting only of the acceptor stem was also functional. These results indicate that the bases essential for FemXWv and AlaRS activities are all located within the acceptor stem of tRNAAla. We therefore focused the following mutagenesis studies on the acceptor stem of tRNAAla although we have not excluded the possibility that additional region of the tRNA might modulate enzyme activity.Figure 4.


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)

Functional RNA molecules in the synthesis of UDP-MurNAc-hexapeptide by AlaRS and FemXWv in the coupled assay. The 76-nt RNA corresponds to the only tRNAAla of E. faecalis. The minimal RNA substrate in the AlaRS-FemXWv coupled assay was a duplex (7 + 11 nt) mimicking the acceptor stem of the tRNAAla.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Functional RNA molecules in the synthesis of UDP-MurNAc-hexapeptide by AlaRS and FemXWv in the coupled assay. The 76-nt RNA corresponds to the only tRNAAla of E. faecalis. The minimal RNA substrate in the AlaRS-FemXWv coupled assay was a duplex (7 + 11 nt) mimicking the acceptor stem of the tRNAAla.
Mentions: Various derivatives of the E. faecalis tRNAAla (Figure 4) were tested as substrates of AlaRS and FemXWv in the coupled assay (Figure 2) for UDP-MurNAc-hexapeptide synthesis. Deletion of the anticodon and pseudouridine arms of tRNAAla resulted in two minihelices, with or without bulged U8, which were both functional in the coupled assay. Formation of UDP-MurNAc-hexapeptide was also obtained with a 24-nt microhelix consisting of the acceptor stem of tRNAAla and a loop of 6 nt. Finally, an RNA duplex consisting only of the acceptor stem was also functional. These results indicate that the bases essential for FemXWv and AlaRS activities are all located within the acceptor stem of tRNAAla. We therefore focused the following mutagenesis studies on the acceptor stem of tRNAAla although we have not excluded the possibility that additional region of the tRNA might modulate enzyme activity.Figure 4.

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