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Journal of Bacteriology, January 2006, p. 269-274, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.269-274.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Two Residues in the Anticodon Recognition Domain of the Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa Are Individually Implicated in the Recognition of tRNAAsn

Dominic Bernard,1,2 Pierre-Marie Akochy,2 David Beaulieu,2 Jacques Lapointe,2 and Paul H. Roy1,2*

Centre de Recherche en Infectiologie, CHUL, 2705 Boul. Laurier RC-709, Ste-Foy, Quebec, Canada G1V 4G2,1 Département de Biochimie et Microbiologie and Centre de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines, Université Laval, Ste-Foy, Quebec, Canada G1K 7P42

Received 4 August 2005/ Accepted 17 October 2005

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNAAsn but by specific tRNA-dependent transamidation of aspartyl-tRNAAsn. This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNAAsp and tRNAAsn with aspartic acid. Recently, it has been shown for the first time in an organism (Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNAAsn but does not participate in Gln-tRNAGln formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNAAsn. Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNAAsp charging over that of tRNAAsn by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNAAsp and tRNAAsn. Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.

* Corresponding author. Mailing address: Centre de Recherche en Infectiologie, CHUL, 2705 Boul. Laurier, RC-709, Sainte-Foy (QC), Canada G1V 4G2. Phone: (418) 654-2705 Fax: (418) 654-2715. E-mail: paul.h.roy{at}crchul.ulaval.ca.

Journal of Bacteriology, January 2006, p. 269-274, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.269-274.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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