Amino acid discrimination by arginyl‐tRNA synthetases as revealed by an examination of natural specificity variants

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‐tRNAArg when using RNA transcribed from the jack bean tRNAACG gene. The corresponding kcat/KM 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 tRNAArg 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.

The influence of identity elements on the aminoacylation of tRNAArg by plant and Escherichia coli arginyl‐tRNA synthetases

“…A similar analysis confirms previous observations [26] reporting that discrimination by the plant enzyme is also sensitive to structural changes in the tRNA (Fig. 11).…”

“…This is toxic to nonproducers, such as soybean, as a result of its incorporation into proteins via misrecognition by the arginyl‐tRNA synthetase [33]. We have previously shown that the structure of the tRNA can influence the discriminating ability of soybean arginyl‐tRNA synthetase [26]. In an approach aiming to determine the role, if any, of tRNA identity elements in amino acid discrimination, we have investigated the structural features that are responsible for tRNA recognition by the arginyl‐tRNA synthetase from the two related higher plants and compared these with previously documented elements in E. coli .…”

“…It should be noted that the jack bean enzyme, which naturally discriminates between arginine and canavanine [26], was found not to canavanylate any of the tRNAs tested. Although kinetic differences and identity element modulations have been detected, the molecular basis distinguishing the discriminating jack bean synthetase from the nondiscriminating soybean enzyme remains to be established.…”

“…The recombinant plant enzymes have been described previously [25,26] [GenBank ID: http://www.ncbi.nlm.nih.gov/nuccore/AM950325 ( C. ensiformis ) and http://www.ncbi.nlm.nih.gov/nuccore/FM209045 ( G. max ), which is identical to the annotated cytoplasmic‐like http://www.ncbi.nlm.nih.gov/nuccore/XM_003529013].…”

“…Aminoacylation with [ 14 C]‐canavanine (50 mCi·mmol −1 ), prepared from l ‐canaline by the barium [ 14 C]‐cyanamide method of Ozinskas and Rosenthal [57], was performed as described previously [26] using a final concentration of 0.4 m m .…”