title: Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.
abstract: Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.
authors.affiliation: Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique, 91128 Palaiseau Cedex, France. codjo@botrytis.polytechnique.fr
authors.forename: Codjo
authors.initials: C
authors.lastname: Hountondji
authors.forename: Christine
authors.initials: C
authors.lastname: Lazennec
authors.forename: Christian
authors.initials: C
authors.lastname: Beauvallet
authors.forename: Philippe
authors.initials: P
authors.lastname: Dessen
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Pernollet
authors.forename: Pierre
authors.initials: P
authors.lastname: Plateau
authors.forename: Sylvain
authors.initials: S
authors.lastname: Blanquet
mesh_terms: D000215:Acylation; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017393:RNA Editing / Q000235:genetics; D012363:RNA, Transfer, Thr / Q000737:chemistry / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000031:analogs & derivatives / Q000378:metabolism; D014637:Valine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012363:RNA, Transfer, Thr; D012366:RNA, Transfer, Val; C011501:methioninyl adenylate; D013912:Threonine; D000249:Adenosine Monophosphate; C018688:valyl adenylate; D008715:Methionine; D014637:Valine-tRNA Ligase; D008239:Lysine; D000409:Alanine