Tài liệu Báo cáo khoa học: Amino acid discrimination by arginyl-tRNA synthetases as revealed by an

Amino acid discrimination by arginyl-tRNA synthetases as

revealed by an examination of natural specificity variants

Gabor L. Igloi and Elfriede Schiefermayr

Institute of Biology, University of Freiburg, Germany

The accuracy of protein biosynthesis is critically

dependent on the fidelity with which aminoacyl-tRNA

synthetases (EC 6.1.1.x) recognize their cognate amino

acid and tRNA substrates [1]. The mechanism(s) by

which the family of aminoacyl-tRNA synthetases

maintains the accuracy of protein biosynthesis has

been the subject of intensive research for some years

[2]. To discriminate between structurally similar amino

acids, whose binding energy difference is insufficient to

guarantee the required distinction [3], some aminoacyl￾tRNA synthetases possess an additional proofreading

or editing activity [4–8] that actively hydrolyses mis￾acylated products. For others that are specific for

structurally idiosyncratic amino acids, no active editing

may be required. In the case of glutamyl- and glutami￾nyl-tRNA synthetases, which together with arginyl￾tRNA synthetase form a subgroup of enzymes that

require tRNA for amino acid activation, the potential

for misrecognition of related amino acids has been

investigated [9–13] and modulated by amino acid

replacements and active site redesign [14]. A mecha￾nism that does not rely on hydrolytic editing but

Keywords

arginyl-tRNA synthetase; L-canavanine;

discrimination; jack bean; soybean

Correspondence

G. L. Igloi, Institute of Biology, University of

Freiburg, Scha¨nzlestr. 1, D-79104 Freiburg,

Germany

Fax: +49 761 203 2745

Tel: +49 761 203 2722

E-mail: [email protected]

(Received 22 September 2008, revised 17

December 2008, accepted 19 December

2008)

doi:10.1111/j.1742-4658.2009.06866.x

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 poly￾peptides. 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 dis￾crimination 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.

Abbreviations

L-Cav, L-canavanine; PCAF, pentacyanoamidoferroate.

FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1307