Glu-Q-tRNA Asp synthetase coded by the yadB gene, a new paralog of aminoacyl-tRNA synthetase that glutamylates tRNA Asp anticodon Mickael Blaise a , Hubert D. Becker a , Jacques Lapointe b , Christian Cambillau c , Richard Giegé a , Daniel Kern a, * a Département Machineries Traductionnelles, UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg cedex, France b Département de Biochimie et Microbiologie, Faculté de Sciences et de Génie, Centre de Recherche sur la Fonction la Structure et l’Ingénierie des Protéines, Université Laval, Québec, Que., Canada G1K7P4 c Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS et Université d’Aix-Marseille I et II, chemin J. Aiguier, 13402 Marseille cedex 20, France Received 31 December 2004; accepted 17 March 2005 Available online 08 April 2005 Abstract Analysis of the completed genome sequences revealed presence in various bacteria of an open reading frame (ORF) encoding a polypep- tide chain presenting important similarities with the catalytic domain of glutamyl-tRNA synthetases but deprived of the C-terminal anticodon- binding domain. This paralog of glutamyl-tRNA synthetases, theYadB protein, activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNA Glu but instead on tRNA Asp . It has been shown that tRNA Asp is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged byYadB. The functional properties ofYadB contrast with those of the canonical glutamyl-tRNA synthetases, which activate Glu only in presence of the cognate tRNA before aminoacylation of the 3′-end of tRNA. Bio- chemical approaches and mass spectrometry investigations revealed thatYadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nucleoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamyl- queuosine. Unstability of the ester bond between the glutamate residue and the cyclopenthene-diol (half-life 7.5 min) explains why until now this modification escaped detection. Among Escherichia coli tRNAs containing queuosine in the wobble position, only tRNA Asp is substrate ofYadB. Sequence comparison reveals a structural mimicry between the anticodon-stem and loop of tRNA Asp and the amino acid acceptor- stem of tRNA Glu .YadB, renamed glutamyl-Q-tRNA Asp synthetase, constitutes the first enzyme structurally related to aminoacyl-tRNA syn- thetases which catalyzes a hypermodification in tRNA, and whose function seems to be conserved among prokaryotes. The discovery of glutamyl-Q-tRNA Asp synthetase breaks down the current paradigm according to which the catalytic domain of aminoacyl-tRNA synthetases recognizes the amino acid acceptor-stem of tRNA and aminoacylates the 3′-terminal ribose. The evolutionary significance of the existence of an aminoacyl-tRNA synthetase paralog dedicated to the hypermodification of a tRNA anticodon will be discussed. © 2005 Elsevier SAS. All rights reserved. Keywords: YadB; Glu-Q-RS; Queuosine; Glutamyl-tRNA synthetase; tRNA aminoacylation; tRNA hypermodification; Paralog Contents 1. Paralogs of aminoacyl-tRNA synthetases ........................................................................................................................ 848 2. Discovery of theYadB protein .......................................................................................................................................... 849 Abbreviations: aaRS, aminoacyl-tRNA synthetase with aa for amino acid (for individual aaRSs, aa is given in the three letter code: e.g. GluRS for glutamyl- tRNA synthetase); Asn synthetase, asparagine synthetase; EF-Tu, elongation factor Tu; Glu-Q, glutamyl-queuosine; Glu-Q-RS, glutamyl-queuosine-tRNA Asp synthetase; GoA, glutamol-adenosine monophosphate; ORF, open reading frame; Q, queuosine; TGT, tRNA-guanine transglycosylase. * Corresponding author. Tel.: +33 3 88 41 70 92; fax: +33 3 88 60 22 18. E-mail address: D.Kern@ibmc.u-strasbg.fr (D. Kern). Biochimie 87 (2005) 847–861 www.elsevier.com/locate/biochi 0300-9084/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2005.03.007