Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multidomain 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 (alpha beta)(2) for A. aeolicus LeuRS, which is present in addition to the alpha beta heterodimer. A. aeolicus (alpha beta)(2) LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alpha beta)(2) enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer up can bind one tRNA(Leu) molecule whereas the heterotetramer (alpha beta)(2) can bind two tRNA(Leu) 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. (c) 2005 Elsevier Inc. All rights reserved.