ABSTRACT
L-canavanine occurs as a toxic non-protein amino acid in more than 1500 leguminous plants. One mechanism of its toxicity is its incorporation into proteins, replacing L-arginine and giving rise to functionally aberrant polypeptides. A comparison between the recombinant arginyl-tRNA synthetases from a canavanine producer (jack bean) and from a related non-producer (soybean) provided an opportunity to study the mechanism that has evolved to discriminate successfully between the proteinogenic amino acid and its non-protein analogue. In contrast to the enzyme from jack bean, the soybean enzyme effectively produced canavanyl-tRNA(Arg) when using RNA transcribed from the jack bean tRNA(ACG) gene. The corresponding k(cat)/K(M) values gave a discrimination factor of 485 for the jack bean enzyme. The arginyl-tRNA synthetase does not possess hydrolytic post-transfer editing activity. In a heterologous system containing either native Escherichia coli tRNA(Arg) or the modification-lacking E. coli transcript RNA, efficient discrimination between L-arginine and L-canavanine by both plant enzymes (but not by the E. coli arginyl-tRNA synthetase) occurred. Thus, interaction of structural features of the tRNA with the enzyme plays a significant role in determining the accuracy of tRNA arginylation. Of the potential amino acid substrates tested, apart from L-canavanine, only L-thioarginine was active in aminoacylation. As it is an equally good substrate for the arginyl-tRNA synthetase from both plants, it is concluded that the higher discriminatory power of the jack bean enzyme towards L-canavanine does not necessarily provide increased protection against analogues in general, but appears to have evolved specifically to avoid auto-toxicity.
Subject(s)
Arginine-tRNA Ligase/chemistry , Genetic Variation , Amino Acid Sequence , Arginine-tRNA Ligase/metabolism , Canavanine/metabolism , DNA, Plant/metabolism , Kinetics , Molecular Sequence Data , RNA, Plant/metabolism , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/metabolism , Substrate Specificity , Transfer RNA AminoacylationABSTRACT
The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.
