Abstract: Aminoacyl-tRNA synthetases (aaRSs) comprise an ancient, diverse enzyme family that catalyzes specific attachment of amino acids to their cognate tRNAs and ensures the accurate translation of the genetic code in the first step of protein synthesis ^[1]. On the basis of conserved sequence and characteristic structural motifs, aaRSs can be divided into two classes (class I and II) with ten members in each class ^[2]. Leucyl-tRNA synthetase (LeuRS) belongs to class Ia aaRSs and the canonical LeuRSs are monomer. Aquifex aeolicus αβ-LeuRS is the only known heterodimeric LeuRS^[3]. By fusion and recombination of the genes encoding the α and β subunits from A. aeolicus αβ-LeuRS and the equivalent amino- and carboxy-terminal parts of E. coli LeuRS (identified as α’ and β’), five monomeric and five heterodimeric LeuRS mutants were obtained. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the β’ part of E. coli LeuRS were not successfully expressed. The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the a subunit of the A.aeolicus enzyme was determined to be deleterious for tRNA charging. Subunit exchange showed that the cross-species-specific recognition of A. aeolicus tRNA^Leu occurs at the a subunit. None of the mixed E.coli-A.aeolicus enzymes were as thermostable as the native αβ-LeuRS. However, the fusion of the α and β peptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme. The editing reactions catalyzed by aaRSs are critical for the faithful protein synthesis by correcting error. We reported that only the isolated editing domain (CP1 domain) of αβ-LeuRS catalyzes the hydrolytic editing of both mischarged tRNA^Leu and minihelix^Leu. Within the domain, we identified a crucial 20-amino-acid motif to editing of CP1 of αβ-LeuRS that confers editing capacity to the inactive isolated CP1 domain of E. coli LeuRS, however the motif did not affect the editing function of αβ-LeuRS. Fusion of the b-subunit of αβ-LeuRS to the E. coli CP1 domain activates its editing function. These results suggest that αβ-LeuRS still carries the basic features from a primitive synthetase molecule.

Key words: Leucyl-tRNA synthetase, tRNALeu, aminoacylation, editing, CP1 domain