Expression of Rat Aspartyl-tRNA Synthetase in Saccharomyces cerevisiae

Abstract

Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities. The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme. We report here the expression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension. The three recombinant enzyme species were purified to homogeneity. They behave as ␣ 2 dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver. Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme. Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site. The two NH 2 -terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements.Aminoacyl-tRNA synthetases are essential enzymes that catalyze a key step during the process of protein biosynthesis: activation of a specific amino acid and the charging of the cognate tRNA (1). On the basis of the comparison of the primary structures of the 20 enzymes from Escherichia coli, it was shown that this family is composed of two phylogenetically distinct protein groups harboring mutually exclusive sets of conserved sequence motifs (2). Accordingly, the topological arrangement of the active site domain, as well as the mode of tRNA:enzyme recognition, are essentially different for these two enzyme classes (3). Class I enzymes are characterized by the two consensus sequences HIGH and KMSKS, which are arranged within a structural fold of the Rossmann type: a six-stranded parallel ␤-sheet. On the other hand, the structural base for the catalytic domain of class II enzymes is a sixstranded anti-parallel ␤-sheet that supports three conserved sequence motifs.Crystallographic studies designate motifs 2 and 3 of class II aminoacyl-tRNA synthetases as essential pieces of their active site (4 -9). This was confirmed by site-directed mutagenesis of presumed key amino acids within these conserved sequences (4, 10 -12). Motif 1 was originally described as a prominent structural element building the subunit interface of class II enzymes that form ␣ 2 dimers. Because the corresponding residues belong to a long ␣-helix and a ␤-strand located at the dimeric interface, it was first believed that motif 1 is a key element of oligomerization. However, recent data suggest that this motif is predominantly inv...

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