Nucleic Acids Research Advance Access originally published online on October 11, 2007
Nucleic Acids Research 2007 35(20):6870-6883; doi:10.1093/nar/gkm778
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Nucleic Acids Research, 2007, Vol. 35, No. 20 6870-6883
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis
1INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, 2Université Pierre et Marie Curie—Paris6, UMR S 872, Paris, F-75006, 3Université Paris Descartes, UMR S 872, Paris, F-75006, 4CNRS UMR 7613, Synthèse, Structure et Fonction de Molécules Bioactives, Paris, F-75005, 5Université Pierre et Marie Curie—Paris6, UMR 7613, Paris, F-75005, 6Muséum National d’Histoire Naturelle, Plateforme de Spectrométrie de Masse et de Protéomique, Département Recherche Développement et Diversité Moléculaire, Paris, F-75005 and 7UMR5154,CNRS-MNHN, Paris, F-75005 France
* To whom correspondence should be addressed. Tel: +33 01 43 25 00 33; Fax: +33 01 43 25 68 12; Email: michel.arthur{at}bhdc.jussieu.fr
Received July 10, 2007. Revised September 13, 2007. Accepted September 17, 2007.
The FemXWv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring L-Ala from Ala-tRNAAla to UDP-MurNAc-pentadepsipeptide. FemXWv 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 FemXWv 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 FemXWv 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 FemXWv activity in agreement with modeling of tRNAAla in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNAAla harboring nucleotide substitutions in the G3–U70 wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemXWv. The different modes of recognition of the acceptor stem indicate that specific inhibition of FemXWv could be achieved by targeting the distal portion of tRNAAla for the design of substrate analogues.
Present address: Antoine P. Maillard, UVHCI, CNRS-EMBL-UJF, Grenoble, France.
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