title: Expression, purification, and characterization of a new heterotetramer structure of leucyl-tRNA synthetase from Aquifex aeolicus in Escherichia coli.
abstract: Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. The non-catalytic domains usually have distinct roles in the aminoacylation reaction. Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) is composed of a separated catalytic site and tRNA anticodon-binding site, which would represent one of the closest relics of the primordial aminoacyl-tRNA synthetase. Moreover, the essential catalytic site residues are split into the two different subunits. In all other class-I aminoacyl-tRNA synthetases, those two functional polypeptides are nowadays fused into a single protein chain. In this work, we report the isolation and the characterization, in Escherichia coli, of a novel oligomeric form (alphabeta)2 for A. aeolicus LeuRS, which is present in addition to the alphabeta heterodimer. A. aeolicus (alphabeta)2 LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alphabeta)2 enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer alphabeta can bind one tRNALeu molecule whereas the heterotetramer (alphabeta)2 can bind two tRNALeu molecules. Active site titration and aminoacylation assays showed that two functional active sites are found per heterotetramer, suggesting that this molecular species might exist and be active in vivo. All those data suggest that the existence of the heterotetramer is certainly not an artifact of overexpression in E. coli.
authors.affiliation: Département de Biologie et Génomique Structurales, UMR7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur Strasbourg, 1 rue Laurent Fries 67404 Illkirch, France.
authors.forename: Natacha
authors.initials: N
authors.lastname: Olieric
authors.forename: Gilbert
authors.initials: G
authors.lastname: Bey
authors.forename: Hélène
authors.initials: H
authors.lastname: Nierengarten
authors.forename: En-Duo
authors.initials: ED
authors.lastname: Wang
authors.forename: Dino
authors.initials: D
authors.lastname: Moras
authors.forename: Gilbert
authors.initials: G
authors.lastname: Eriani
authors.forename: Jean
authors.initials: J
authors.lastname: Cavarelli
mesh_terms: D049148:Aminoacylation; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006091:Gram-Negative Chemolithotrophic Bacteria / Q000201:enzymology* / Q000235:genetics; D007935:Leucine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012343:RNA, Transfer; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D012343:RNA, Transfer; D007935:Leucine-tRNA Ligase