Amino acid discrimination by arginyl-tRNA synthetases as revealed by an examination of natural specificity variants.
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.
Igloi
Gabor L
GL
Institute of Biology, University of Freiburg, Freiburg, Germany. igloi@biologie.uni-freiburg.de
Schiefermayr
Elfriede
E
D000595:Amino Acid Sequence; D001126:Arginine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D002172:Canavanine / Q000378:metabolism; D018744:DNA, Plant / Q000378:metabolism; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D018749:RNA, Plant / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D046249:Transfer RNA Aminoacylation
D018744:DNA, Plant; D018749:RNA, Plant; D012346:RNA, Transfer, Amino Acyl; D002172:Canavanine; D001126:Arginine-tRNA Ligase