Tài liệu Báo cáo khoa học: Amino acid discrimination by arginyl-tRNA synthetases as revealed by an examination of natural specificity variants doc

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 Keywords arginyl-tRNA synthetase; L-canavanine; discrimination; jack bean; soybean Correspondence G L Igloi, Institute of Biology, University of Freiburg, Schanzlestr 1, D-79104 Freiburg, ¨ Germany Fax: +49 761 203 2745 Tel: +49 761 203 2722 E-mail: igloi@biologie.uni-freiburg.de (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 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 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 aminoacyltRNA synthetases possess an additional proofreading or editing activity [4–8] that actively hydrolyses misacylated products For others that are specific for structurally idiosyncratic amino acids, no active editing may be required In the case of glutamyl- and glutaminyl-tRNA synthetases, which together with arginyltRNA 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 mechanism that does not rely on hydrolytic editing but Abbreviations L-Cav, L-canavanine; PCAF, pentacyanoamidoferroate FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1307 Arginyl-tRNA synthetase amino acid discrimination G L Igloi and E Schiefermayr resembles an induced fit type of substrate selection, including the participation of tRNA structural features, has been proposed [14] The specificity of arginyl-tRNA synthetase (EC 6.1.1.19) towards amino acids for which a similar discriminatory mechanism may be required has not been studied systematically Research regarding the accuracy of protein biosynthesis has, in the past, been largely devoted to prokaryotes and lower eukaryotes (yeast) With isolated exceptions in the early literature, aminoacyl-tRNA synthetases from plants, which must not only discriminate between the 20 common amino acids but must also contend with related potentially toxic natural analogues [15,16], have been ignored This challenge faced by plants offers a natural alternative to targeted mutagenesis or rational redesign of the active site of the enzymes to elucidate the mechanism by which fidelity of amino acid selection is maintained We have focused our attention on a pair of species-specific enzyme variants, one of which is said to be evolutionarily adapted to reject a naturally occurring toxic arginine analogue [17], while the other lacks this ability l-Canavanine [18,19] [l-2-amino-4-(guanidinooxy)butyric acid], the guanidino-oxy structural analogue of arginine (Fig 1) 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 [20–22] A comparison between the recombinant arginyl-tRNA synthetases from a canavanine producer (jack bean, Canavalia ensiformis) and from a related non-producer (soybean, Glycine max) provides an opportunity to gain insight into the mechanism of amino acid recognition in the arginine system NH2 HN O NH OH NH2 L-Arginine NH2 H2N N O O OH NH2 L-Canavanine Fig Structures of L-canavanine 1308 L-arginine and its guanidinooxy analogue, Results On the basis of the annotated Arabidopsis genome, we established the cDNA sequence of the argS gene of jack bean (accession number AM950325) [23] and of soybean (accession number FM209045) The derived proteins comprise 597 (soybean) and 595 (jack bean) amino acids, with molecular masses of 68.2 and 67.4 kDa, respectively The genes for arginyl-tRNA synthetase from jack bean and soybean were cloned into the bacterial expression vector pET32a and transformed into Escherichia coli BL21 cells Despite their sequence similarity (Fig 2), the enzyme from soybean proved much more resistant to soluble expression than the one from jack bean [23] The yield from jack bean (10 mgỈL)1 cell culture) compares with 1.2 mgỈL)1 culture for soybean Removal of the His-tag ⁄ thioredoxin fusion by cleavage at the enterokinase site provided by the vector was unsuccessful However, the thrombin site, located 30 amino acids upstream of the native synthetase sequence, was accessible to proteolysis A predicted internal thrombin site (position 130 of the native protein) in the soybean arginyl-tRNA synthetase was not targeted by this protease The position of the cleavage was confirmed by N-terminal protein sequencing The results reported here were obtained using thrombin-treated preparations of arginyl-tRNA synthetases that retained a 3.2 kDa N-terminal extension compared to the native enzyme Sequence analysis of the tRNAArgACG gene from Canavalia ensiformis established its identity to the Arabidopsis sequence (accession number NR_023294) The subsequent appearance in the NCBI trace archives of a sequence corresponding to the gene of soybean tRNAACG (accession number gnl|ti|1583039205) confirmed its similarity to the jack bean sequence with a single base difference from A (jackbean) to G (soybean) at position 37 The chemically synthesized gene for tRNAArgACG from jack bean was cloned, and the full-length tRNA was generated by in vitro transcription The transcript could be aminoacylated with arginine to a level of approximately 0.05 pmol amino acid ⁄ pmol tRNA The corresponding soybean transcript had an arginine acceptance level of approximately 0.1 pmol amino acid ⁄ pmol tRNA As is the case for arginyl-tRNA synthetases from other sources [24–26], the pyrophosphate exchange reaction is absolutely dependent on the presence of aminoacylatable tRNA Periodate-oxidized tRNA, which has been shown to be inactive in aminoacylation, did not stimulate pyrophosphate exchange (Fig 3) The tRNA concentration dependence of this reaction gives a KM value that is equivalent to that of FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS G L Igloi and E Schiefermayr Arginyl-tRNA synthetase amino acid discrimination Fig Alignment of derived arginyl-tRNA synthetase primary structures from jack bean (Ce, Canavalia ensiformis), soybean (Gm, Glycine max) and yeast (Sc, Saccharomyces cerevisiae) Shading in black indicates identity in all three sequences; shading in grey indicates identity in two sequences PPi exchange in to ATP (Pmol) 120 100 80 60 40 20 –20 10 12 14 16 Time (min) Fig Dependence of the pyrophosphate exchange reaction on tRNA The pyrophosphate exchange reaction was carried out in the absence ( ) or the presence of lM ( ) or 30 lM (r) transcript tRNA or 12 lM (d) periodate-oxidized jack bean transcript tRNA using jack bean arginyl-tRNA synthetase PPi, tetrasodium pyrophosphate tRNA as measured by aminoacylation (data not shown) Using either [14C]-canavanine in the conventional aminoacylation assay, or unlabelled canavanine together with [32P]-labelled jack bean transcript tRNA, it was observed that the soybean enzyme effectively transferred this amino acid to the transcript tRNA, but it was a much poorer substrate for the jack bean enzyme (Fig 4, inset) To examine whether the arginyl-tRNA synthetases from the two plants show different specificities towards other arginine analogues, the [32P]-labelled tRNA assay was used to screen a selection of amino acids, including ones that have previously been shown not to be substrates for the enzyme from other sources l-thiocitrulline and the naturally occurring l-homoarginine, l-citrulline, l-homocitrulline and l-albizziine (l-2-amino-3-ureidopropanoic acid) were, at mm concentration, if at all, extremely poor substrates for both plant enzymes (Fig 4), and FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1309 Arg Cav Arg G L Igloi and E Schiefermayr Cav Arginyl-tRNA synthetase amino acid discrimination Aminoacyl-A76 100 tRNA 80 Origin 40 20 So yb e a n 60 Jack bean Aminoacylation (% arginylation) 120 NH H2N OH O NH2 O OH OH NH2 O OH S H 2N NH NH O NH2 NH2 NH NH i ne O iine NH2 va n a na z lbiz NH2 OH L -C L -A ine e ysi n L -L lline itr u hioc L -T ne rulli ocit om L -H lline utru L -C ine rgin hioa L -T ne gini oar om rgin L -H L -A OH O Soybean enzyme Jack bean enzyme OH OH O O S NH2 H2N NH NH NH2 O NH2 NH O NH2 NH NH2 OH O NH2 O N H2N H2N NH2 O NH2 Fig Quantitative comparison of amino acid utilization by the plant arginyl-tRNA synthetases The aminoacylation level attained in the presence of L-arginine was compared to that in the presence of mM of the analogue indicated, using [32P]-labelled jack bean transcript tRNA Inset: Activity of arginyl-tRNA synthetase from jack bean and soybean with L-canavanine, under the above conditions Aminoacylation is characterized by the liberation of labelled aminoacyl-A76 after nuclease P1 treatment l-lysine charging was barely detectable The synthetic arginine analogue, l-thioarginine, recently introduced as a substrate for arginase [27], was extensively transferred to tRNA by both enzymes (KM for soybean 56 lm; KM for jack bean 81 lm) In order to quantify the discrimination exhibited by the plant enzymes with respect to canavanine, kinetic parameters for aminoacylation were determined using the tRNA transcript derived from the jack bean gene Radioactive canavanine was efficiently transferred to the plant tRNA transcript by the arginyl-tRNA synthetase from soybean In this case, the kinetic parameters correspond to a discrimination factor, (kcat ⁄ KM)Arg ⁄ (kcat ⁄ KM)Cav, of 44 (Table 1) A similar factor was obtained when assayed with non-radioactive canavanine using the [32P]-labelled tRNA assay [28] For the jack bean enzyme, a distinct discrimination between 1310 arginine and canavanine for aminoacylation of the plant tRNA transcript was observed when using [14C]-canavanine At 0.4 mm canavanine, less than 10% of the tRNA was aminoacylated compared to arginine transfer This low but significant level of mischarging is the result of a relatively modest degree of discrimination Using the sensitive [32P]-labelled tRNA assay and higher concentrations of canavanine, a KM for this substrate of 1.3 mm was determined, and the relative magnitude of the kcat ⁄ KM parameters for arginine and canavanine charging revealed a discrimination factor of 485; a factor of 10 greater than for the soybean enzyme (Table 1) The discrimination based on catalytic efficiency may in itself be insufficient to guarantee survival of the canavanine-producing plant An additional classic post-transfer proofreading mechanism [7,29] would require the rapid deacylation of Cav-tRNAArg by the FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS G L Igloi and E Schiefermayr Arginyl-tRNA synthetase amino acid discrimination Table Quantification of discrimination between L-arginine and L-canavanine using jack bean transcript tRNA Assays were based on the aminoacylation reaction using either [14C]-labelled amino acids or [32P]-labelled tRNA Assay method Aminoacylation of transcript tRNA with [14C]-labelled amino acid Aminoacylation of [32P]-labelled transcript tRNA Arg Arg Cav Cav Source of enzyme KM (lM) kcat ⁄ KM (M)1Ỉmin)1) KM (lM) kcat ⁄ KM (M)1Ỉmin)1) KM (lM) kcat ⁄ KM (M)1Ỉmin)1) KM (lM) kcat ⁄ KM (M)1Ỉmin)1) Discrimination factor(kcat ⁄ KM)Arg ⁄ (kcat ⁄ KM)Cav Jack bean Soybean 19.6 2.2 1.0 9.2 NDa 45.3 ND 0.27 7.8 3.0 0.82 3.2 1320 45.4 0.0017 0.055 482 34 ([14C]-labelled amino acid); 58 ([32P]-labelled tRNA) a ND, not determined because of the impracticality of using large amounts of [14C]-Cav jack bean enzyme Cav-tRNAArg was prepared by canavanylation of the jack bean tRNA transcript using arginyl-tRNA synthetase from soybean The stability of the isolated charged tRNA was compared in the presence of arginyl-tRNA synthetase from soybean or jack bean (Fig 5) The first-order decay curves correspond to a half life of only approximately for Cav-tRNAArg even in the absence of either enzyme In contrast, the half life of Arg-tRNAArg is 46 Addition of arginyl-tRNA synthetase from jack bean does not further decrease the stability of the canavanylated species The role of tRNA as a cofactor for aminoacylation in those aminoacyl-tRNA synthetases that require tRNA for amino acid activation is well documented [9], and the determinants within the tRNA that are 120 required for arginine activation by a mammalian enzyme have been established using various constructs, including tRNA chimeras comprising domains from yeast [26] If or how these structural elements are involved in amino acid discrimination was not specified Using the pair of plant arginyl-tRNA synthetases characterized here, it is possible to investigate how alterations in the tRNA structure manifest themselves in terms of misaminoacylation As a first approach, we screened a number of heterologous tRNA ⁄ enzyme pairs for aminoacylation tRNAs from a number of sources, when compared to the activity with E coli arginyl-tRNA synthetase, proved to be arginylated by the plant enzymes (Fig 6) In absolute terms, transcripts of tRNA genes were poorly arginylated by their respective enzymes (Table 2) Remarkably, the soybean enzyme was no longer able to attach canavanine to E coli tRNAArgACG (Fig 7) despite the fact that 140.00 Arginylation level (% attained with E.c oli enzyme) Aminoacyl-tRNA remaining (%) 100 80 60 40 20 0 10 15 20 Time (min) 25 30 120.00 100.00 60.00 40.00 20.00 0.00 35 Fig Stability of canavanyl-tRNA Jack bean transcript tRNAArg that had been aminoacylated with [14C]-L-canavanine was incubated in the absence of enzyme ( ), or in the presence of jack bean (d) or soybean (,) arginyl-tRNA synthetase, and the amount of aminoacyl-tRNA remaining after a given time was quantified Alternatively, [14C]-L-arginyl-tRNA was incubated in the absence of enzyme ()) 80.00 E.coli E.coli Bovine Jack bean Soybean Wheat native transcript transcript transcript germ total liver total tRNA-Arg tRNA tRNA Source of tRNA Fig Interspecies arginylation tRNA from the sources indicated were arginylated in the presence of arginyl-tRNA synthetase from jack bean (diagonal shading) or soybean (vertical shading), and the level of charging was compared with that in the presence of the E coli enzyme FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1311 Arginyl-tRNA synthetase amino acid discrimination G L Igloi and E Schiefermayr Table Arginine acceptance by homologous and heterologous tRNAs Arginine acceptance by native E coli tRNAArg was compared with that of modification-lacking tRNA transcripts using E coli or plant arginyl-tRNA synthetases ND, not determined E coli native tRNAArg E coli transcript Jack bean transcript Soybean transcript Source of enzyme Aminoacylation (pmol ArgỈpmol)1 tRNA) KM (lM) Aminoacylation (pmol ArgỈpmol)1 tRNA) KM (lM) Aminoacylation (pmol ArgỈpmol)1 tRNA) KM (lM) Aminoacylation (pmol ArgỈpmol)1 tRNA) KM (lM) E coli Jack bean Soybean 0.64 0.61 0.46 ND 0.86 1.5 0.07 0.05 0.06 ND 1.2 1.1 0.1 0.06 0.05 ND ND ND 0.11 0.09 0.11 ND ND ND E coli tRNA is a good substrate for arginylation The presence of E coli tRNA, irrespective of whether native or the modification-lacking transcript, caused ‘evolution’ of a discriminatory soybean enzyme that could, in contrast to the E coli enzyme, reject canavanylation as efficiently as the jack bean enzyme The jack bean enzyme did not charge either its cognate tRNA or the transcript corresponding to the soybean sequence with canavanine Discussion The evidence that the arginyl-tRNA synthetase of a canavanine producer, e.g jack bean (Canavalia ensiformis), can discriminate between l-arginine and its analogue is indirect It relies on the observation that jack bean plants injected with radioactive l-canavanine not incorporate the label into their proteins, compared to soybean plants, which [30] In a previous study, ‘somewhat indefinite’ conclusions regarding activation of canavanine by the arginyl-tRNA synthetase from Canavanylation (% Arg incorportation) 100 90 80 70 60 50 40 30 20 10 Jack bean transcript Soybean transcript Wheat germ Bovine liver E.coli native total tRNA total tRNA tRNA-Arg E.coli transcript Fig Comparison of canavanine incorporation The amount of L-canavanine transferred by arginyl-tRNA synthetase from E coli (waved shading), jack bean (vertical dashes) and soybean (diagonal shading) to the tRNA species indicated was quantified using 0.4 mM [14C]-L-canavanine relative to the corresponding arginine incorporation 1312 Canavalia ensiformis [17] were reported However, the pyrophosphate exchange assay, in the absence of the absolutely required tRNA [24], was used to study substrate specificity The apparent arginine activation described may be due to a co-purified lysyl-tRNA synthetase (as characterized in the same publication), that does not require tRNA for pyrophosphate exchange and can accept arginine [31,32] While the subsequent discovery of a corrective proofreading activity of several aminoacyl-tRNA synthetases [6–8] provides a reasonable basis for assuming an evolution of a discriminating function by the jack bean enzyme, we considered that investigation of a natural, discriminating ⁄ non-discriminating pair of enzymes would provide further insight into this process The translated gene sequences proved to be 85% identical to each other but had only 25% identity to the yeast enzyme, the only eukaryotic arginyl-tRNA synthetase whose 3D structure has been elucidated to date [33] Despite this limited similarity and the fact that arginyltRNA synthetases from fungi are considered to belong to a distinct class [34], certain features that have been identified in yeast as being involved in substrate binding [35] are conserved in the plant enzymes In the case of tRNA recognition, G(483:Y), which is part of the so-called X loop and is said to form a molecular switch [33], is conserved (Fig 2) [We refer here to comprises the one-letter amino acid followed by its position in the sequence of the organisms whose name is abbreviated after the colon, i.e Y, yeast; C, Canavalia ensiformis (jack bean); G, Glycine max (soybean)] Other residues participating in hydrophobic interactions, such as F(109:Y) and L(70:Y), are also conserved, and may align with F(100:C), F(102:G) and L(59:C), L(61:G), respectively On the other hand, R(66:Y), R(75:Y) and K(102:Y) not align with any charged residues in the jack bean or soybean, leaving one to speculate on the source of the interaction with the sugar–phosphate backbone Correct positioning of the essential Ade76 of the tRNA has been ascribed to residues E(294:Y), Y(347:Y) and N(153:Y) [35], all of which are conserved at corresponding positions in jack FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS G L Igloi and E Schiefermayr bean and soybean When binding of arginine in the presence of tRNA was investigated, some changes in the binding architecture were observed [35], in that N(153:Y), in addition to interacting with the a-carboxylate, also associates with the 2¢O of Ade76 Similarly, Y(347:Y) recognizes the guanidinium g-N but also comes into contact with the adenosine ring of Ade76 There is a general consensus that tRNA binding is not required for arginine binding [33], although arginine binding is a prerequisite for correct positioning of the CCA end, mediated through movement of a conserved tyrosine [Y(347:Y)] to a different conformation [26], allowing ATP to bind productively Although arginine and canavanine are stereochemically similar, the presence of the oxygen atom in canavanine dramatically influences the pKa of the guanidine group, lowering the value from 12.5 by more than pKa units [36,37], locking the molecule in an imino-oxy tautomer (Fig 1) and resulting in a largely uncharged side chain at physiological pH Transcripts derived from the sequences of the tRNAArgACG genes from jack bean and soybean were arginylated to only 6–10% of the theoretical acceptance by the arginyl-tRNA synthetases from both jack bean and soybean, although the KM for the jack bean tRNA resembles that of native tRNA (Table 2) In general, the efficiency of transcript aminoacylation may be close to 100% [38,39] but can be substantially less [40–42] It has been proposed that the presence of base modifications leads to reduced flexibility of the tRNA molecule [38], whereas G:U base pairs are responsible for the tRNA flexibility required for arginylation in a mammalian system [26] Despite the low level of arginine acceptance by the transcripts, there was a clear distinction between the two enzymes when it came to canavanine incorporation The enzyme from jack bean produces only low levels of canavanyl-tRNA with both its cognate and the soybean tRNA In contrast, the soybean enzyme effectively linked the analogue to both plant tRNAs Examination of the kinetics of the reaction revealed a significantly higher affinity of the soybean synthetase for canavanine (69 lm) compared with that of the jack bean enzyme (1.3 mm), and the corresponding kcat ⁄ KM values result in discrimination factors of approximately 40 and 485 for the respective enzymes However, in a heterologous system using either native E coli tRNAArgICG or a transcript of the corresponding gene, we observed how the structure of the tRNA itself can modulate the efficiency of discrimination Whereas these tRNAs are arginylated efficiently by the synthetases from E coli, jack bean and soybean, and although canavanylation to a high level is achieved by the E coli enzyme, the soybean enzyme Arginyl-tRNA synthetase amino acid discrimination reveals a discriminatory ability that has characteristics approaching those of the jack bean enzyme In view of the distinct role of conformational changes that accompany the catalytic cycle of the mammalian enzyme [26], one should consider the possibility that the amino acid-dependent positioning of the tRNA (or the CCA end) in a functional configuration, mediated by global conformational changes in the protein, could be a further factor in preventing the formation of misacylated tRNA For arginyl-tRNA synthetase, rearrangement of the enzyme active site appears to rely on additional discriminatory elements within the tRNA structure to ensure accurate formation of aminoacyltRNA This is reminiscent of the glutamyl- and glutaminyl-tRNA synthetases of E coli For glutamyl-tRNA synthetase, the presence of tRNA eliminates non-specific binding of d-glutamic acid and l-aspartic acid to the enzyme [9,10] Detailed analysis of glutaminyl-tRNA synthetase has led to the proposal of an induced-fit type of active site rearrangement that plays a role in enzyme specificity [11–13], and the concept of discriminatory elements in tRNA that participate in amino acid selection has been proposed [14] It would then be consistent with our observations for jack bean tRNAArg to trigger an active site rearrangement in the jack bean enzyme that provides the means to enhance amino acid discrimination The fact that the association of the same tRNA with the soybean enzyme promotes both arginylation and canavanylation, while in the heterologous system the soybean enzyme is unable to canavanylate the E coli tRNA, is an indication of the subtlety of this structural interplay, that requires further investigation An additional classic post-transfer proofreading mechanism [6,29], that is not observed in the glutamine or glutamic acid systems [9,12] but that would enhance the overall accuracy, would require rapid, specific deacylation of Cav-tRNAArg by the jack bean enzyme Cav-tRNAArg prepared by canavanylation of the jack bean tRNA transcript using arginyl-tRNA synthetase from soybean is highly unstable, being rapidly hydrolysed at neutral pH even in the absence of added enzyme This instability (half life of approximately min) compared to arginyl-tRNA (half life of 46 min) may be attributed to the electronic charge distribution of the canavanyl ester that promotes rapid degradation However, as no additional enzyme-specific destabilization was observed, post-transfer hydrolytic proofreading may be ruled out The low discrimination factor achieved by the soybean enzyme leads to efficient canavanylation of tRNAArg in vitro and incorporation of this allelochemical into proteins in vivo [30,43] However, the several hundred-fold discrimination measured for the jack FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1313 Arginyl-tRNA synthetase amino acid discrimination G L Igloi and E Schiefermayr bean enzyme is considerably lower than the factor of 104 normally expected from systems that rely on an active proofreading process to correct misrecognized substrates [8] Nevertheless, physiological evidence indicates that canavanine producers not incorporate this toxic analogue into their proteins A discrimination factor between leucine and isoleucine of similarly modest magnitude (approximately 600) has been described for leucyl-tRNA synthetase from E coli [44] In that case, it was suggested that an evolutionary balance between catalytic efficiency and specificity can lead to sacrifices in both these parameters This may be reflected in the 5–10-fold reduced relative kcat ⁄ KM for the jack bean enzyme compared to the soybean synthetase Additionally, to what extent low levels of mischarged tRNA can be tolerated [45] or other in vivo processes such as discrimination at the stage of elongation factor ⁄ aminoacyl-tRNA complex formation [2,46,47], competition between various cellular levels of the amino acids, or metabolic processes competing for canavanine utilization [48] contribute to the overall avoidance of auto-toxicity remains to be seen The ability of the jack bean enzyme to distinguish between the secondary metabolite canavanine and its intended substrate arginine appears to have evolved specifically Other arginine analogues such as l-ornithine, l-a-amino-c-guanidinobutyric acid, l-citrulline, l-homocitrulline or l-homoarginine have been assessed as substrates for arginyl-tRNA synthetases from various non-plant sources [49–51], and have at best been weak inhibitors but are generally not incorporated into proteins [20,52] Of the potential substrates that we have tested, apart from l-canavanine, only l-thioarginine [27] was activated significantly In contrast to l-canavanine, it is the bridging N of the guanidine group that is replaced by the heteroatom in l-thioarginine, locking the guanidino nitrogens into the arginine-like tautomeric form As we have shown that l-thioarginine is an effective and equally good substrate for the arginyltRNA synthetases from both plants, we conclude that the higher discriminatory power of the jack bean enzyme towards canavanine is a specific evolutionary property that may not necessarily provide increased protection against analogues in general Experimental procedures Primers were designed using oligo 5.0 (MedProbe, Oslo, Norway) or gap4 of the Staden Package [53], synthesized using an ABI3948 nucleic acid synthesis and purification system (Applied Biosystems, Foster City, CA, USA) by the Freiburg Institute of Biology core facility DNA sequence analysis was performed using BigDye version 1.1 chemicals 1314 (Applied Biosystems) in combination with an ABI Prism 310 genetic analyser Contigs were assembled using the Staden Package [53] Native nucleotidyl transferase from yeast originated from the stocks of H Sternbach (formerly MaxPlanck-Institute, Gottingen), while that from E coli in ă recombinant form was provided by A Weiner (University of Washington School of Medicine, Seattle, WA, USA) [14C]l-arginine (12.8 GBqỈmmol)1) was purchased from PerkinElmer (Waltham, MA, USA) l-homoarginine, l-citrulline and l-thiocitrulline were obtained from Acros Organics (Geel, Belgium) The source of other chemicals was as follows: l-homocitrulline (Advanced Asymmetrics, Millstadt, IL, USA), l-albizziine (2-amino-3-ureidopropanoic acid) (Bachem, Bubendorf, Switzerland), l-canavanine (Sigma, Munich, Germany) and l-thioarginine (l-2-amino-5-isothioureidovaleric acid) (Cayman Chemical, Tallinn, Estonia) An extract from E coli, active for aminoacylation, was obtained by depleting an S30 bacterial supernatant of endogenous nucleic acids by fractionation on a DEAE-cellulose column Bulk tRNAs from wheat germ and from calf liver were purchased from Sigma E coli tRNA enriched in tRNAArgACG to an arginine acceptance of 760 pmol ⁄ A260 was obtained from an expression construct provided by G Eriani (Institut ´ de Biologie Moleculaire et Cellulaire, Strasbourg, France) and E.-D Wang (Shanghai Institutes for Biological Sciences, China) [54] DNA and RNA isolation Total RNA was isolated from 100 to 200 mg leaf tissue from to 4-week-old soybean (Soybean UK, Southampton, UK) or jack bean (Sigma) plants using RNeasy plant mini kits (Qiagen, Hilden, Germany) cDNA was prepared using a T17-mer and Superscript reverse transcriptase (Invitrogen, Karlsruhe, Germany) Sequences were identified by blast comparison (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) Gene for arginyl-tRNA synthetase The gene for the enzyme from jack bean has been characterized recently [23] (accession number AM950325) For the soybean sequence (accession number FM209045), the translated cDNA sequence of Arabidopsis arginyl-tRNA synthetase (accession numbers NM_118763 and NM_ 105324) was aligned with the corresponding sequences in other eukaryotes Soybean EST fragments mined from the databases were compiled to identify conserved regions, reverse-translated and used to design primers for cDNA amplification The longest PCR fragment obtained by combining the gene-specific probes with a T17 primer and whose sequence could be identified as being that of arginyltRNA synthetase was used to generate primers for stepwise 5¢ RACE elongation of the sequence [55] PCR products were purified using Montage cartridges (Millipore, Eschborn, Germany) FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS G L Igloi and E Schiefermayr Gene for tRNAArgACG from jack bean Total tRNA from jack bean was obtained from cellular RNA by extraction with m NaCl, and purified by DEAESephadex chromatography as described previously [56] tRNA (1 lg) was ligated to 20 pmol of a 5¢-phosphorylated, 3¢-periodate-oxidized hybrid RNA ⁄ DNA oligonucleotide [5¢p-rCrCd(CCTCCTTTTATTcactggccgtcgttttacTC)r Aox] synthesized on an ABI 394 DNA ⁄ RNA synthesizer (Applied Biosystems) The oligonucleotide was designed to permit efficient ligation through its 5¢-ribonucleotides, enable the use of the universal M13 primer for reverse transcription (binding region in lower case), and prevent selfligation after periodate oxidation of the 3¢-terminal ribose Ligation was performed in HCC buffer [57] using 50 units of T4 RNA ligase (GE Healthcare, Munich, Germany) in a total volume of 50 lL For reverse transcription, lL of the ligation product was annealed to pmol of universal M13 primer, and the reaction was performed under standard conditions using 15 units of Thermoscript reverse transcriptase (Invitrogen) After incubation for h at 56 °C, the reaction was terminated by heating to 85 °C for min, followed by RNase H treatment (GE Healthcare) for 20 at 37 °C The gene specific for tRNAArg was amplified using the universal M13 primer, which binds to the 3¢ tail of the RNA, and an 18-mer based on the 5¢ terminus of tRNAArgACG from Arabidopsis (accession number AT1G13010) The amplicon was sequenced using the M13 primer to give the Canavalia ensiformis 3¢-terminal 55-base sequence The remaining 5¢ region was assembled taking into account conserved D-loop bases and the base-pairing requirement of the D-loop and acceptor stems, while bearing in mind that none of the 14 plant tRNAArgACG sequences available in the databases possess a G:U base pair in the acceptor stem (data not shown) Protein expression Cloning and bacterial expression of the His-tagged soybean enzyme was performed as described for jack bean [23] Thrombin treatment to remove the His tag was performed as described previously [23] In the case of the soybean enzyme, an additional cleaning step comprised adsorption on Source15Q (GE Healthcare) followed by an 80 mm NaCl wash and elution at 0.3 m NaCl The homogeneity of the preparation was monitored by SDS–PAGE, and the identity of the protein was confirmed by N-terminal sequencing In vitro transcription The genes for jack bean and soybean tRNAArgACG were synthesized as a single-stranded oligonucleotide and then amplified by PCR using appropriate primers bearing a T7 promoter extension Transcription at a 0.5 mL scale was Arginyl-tRNA synthetase amino acid discrimination performed in T7 RNA polymerase buffer (40 mm Tris ⁄ HCl pH 8, 12 mm MgCl2, mm dithiothreitol, mm spermidine HCl, 4% polyethylene glycol 8000, 0.002% Triton X-100), mm NTP, 20 mm GMP, 0.1 units of inorganic pyrophosphatase (Sigma), 0.7 nmol template DNA, and 52 nm T7 RNA polymerase prepared from the recombinant pAR1219 expression plasmid [58] Incubation was performed for h at 37 °C, and was followed by purification by NAP-5 gel filtration (GE Healthcare), phenol extraction and ethanol precipitation Its homogeneity, as determined by denaturing polyacrylamide gel electrophoresis, was greater than 80% The tRNA was refolded by heating to 70 °C in water, followed by slow cooling in the presence of 25 mm Tris-HCl, pH 7.5, 250 mm NaCl, mm MgCl2 Colorimetric detection of canavanine Canavanine detection and quantification were achieved by following its colour reaction with pentacyanoamidoferroate (PCAF) (ICN Biomedicals, Aurora, OH, USA) [59] using an ND-1000 photometer (NanoDrop Technologies, Wilmington, DE, USA) To the canavanine-containing sample in 10 lL was added 10 lL of 200 mm potassium phosphate pH 7.5, lL 1% potassium persulphate and lL 1% PCAF in water The colour was allowed to develop for 40 at room temperature and the absorbance at 530 nm was measured Preparation of L-canaline Synthesis of radioactive canavanine from l-canaline was based on a previously described procedure [60] using [14C]cyanamide as a guanylating reagent As l-canaline is no longer commercially available, l-canavanine sulphate was converted to l-canaline by arginase treatment, essentially as described previously [61] The arginase required for this was obtained as a crude extract from the leaves of Canavalia brasiliensis The extract enriched in arginase was used immediately for preparative-scale conversion of canavanine to canaline Canaline was recovered from the reaction mixture as its picrate salt, and converted to the free base as described previously [61] Elemental analysis indicated C 35.81% (calculated 35.82%), H 7.66% (calculated 7.51%), N 19.43% (calculated 20.88%) Canaline was stored desiccated at )20 °C Synthesis of [14C]-L-canavanine [Guanidino-14C]-l-canavanine was synthesized essentially as described previously [60] from 46 lmol canaline free base and mCi barium [14C]-cyanamide (57.5 mCiỈmmol)1, 34.8 mmol; Moravek, Brea, CA, USA) The required pH adjustments were made using a micro pH electrode (Metrohm, Filderstadt, Germany) Analysis by TLC on FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1315 Arginyl-tRNA synthetase amino acid discrimination G L Igloi and E Schiefermayr silica (EtOH : AcOH : H2O, 65 : : 34) gave a single PCAF-reactive spot with 95% isotopic homogeneity, and the canavanine-specific PCAF reaction showed the presence of canavanine at 20 mm concentration containing a total of 1.4 mCi radioactivity (50 mCiỈmmol)1) The stock solution was stored at )70 °C in the presence of 2% ethanol Pyrophosphate exchange Pyrophosphate exchange was monitored at 30 °C in the presence of 50 mm Hepes ⁄ KOH pH 7.5, 10 mm MgCl2, 1.5 mm ATP, 80 lm [32P]-tetrasodium pyrophosphate (Perkin-Elmer; specific activity in assay 5–10 cpmỈpmol)1), together with amino acid, tRNA and enzyme in 50 lL reactions Radioactivity incorporated into ATP was quantified by spotting 10 lL aliquots of the reaction onto 25 mm diameter charcoal-impregnated filters (Type 69K) (Munktell, Barenstein, Germany) [62] Filters were washed for ă 10 in 1.5% perchloric acid ⁄ 40 mm pyrophosphate, followed by rinsing with water, before being dried under infrared lamps Scintillation counting was performed using Rotiszint (Roth, Karlsruhe, Germany) Aminoacylation Aminoacylation was performed at 30 °C in a volume of 50 lL containing 50 mm Hepes ⁄ KOH pH 7.5, 10 mm MgCl2, mm ATP and the appropriate amount of [14C]amino acid, tRNA and arginyl-tRNA synthetase Amino acid incorporation was followed using MM filter discs (Whatman, Dassel, Germany) that had been pretreated with 50 lL 5% trichloroacetic acid (to reduce non-specific background, particularly when using [14C]-canavanine) and dried Aliquots were spotted onto the discs which were then washed with two changes of 5% trichloroacetic acid and once with ethanol (10 each), before being dried and quantified by scintillation counting Preparative aminoacylation reactions, scaled to 100 lL, were allowed to reach a plateau, rapidly extracted with phenol, and the aminoacylated tRNA was collected by ethanol precipitation at pH 4.8 Alternatively, the procedure described by Wolfson and Uhlenbeck [28] to detect the incorporation of unlabelled amino acids into [32P]-labelled tRNA was used The tRNA transcript was labelled with [a-32P]-ATP (111 TBqỈmmol)1) (Perkin-Elmer) in the presence of yeast or E coli tRNA nucleotidyl transferase Approximately 0.1 lCi tRNA and 0.35 nmol unlabelled tRNA was aminoacylated in a 10 lL total volume containing 50 mm Hepes pH 7.5, 10 mm MgCl2 and 2.5 mm ATP together with aminoacyl-tRNA synthetase and amino acids as indicated in the text Aliquots (1 lL) were transferred to lL 200 mm NaOAc pH containing 0.4 units of nuclease P1 (Roche, Mannheim, Germany) Digestion proceeded at room temperature for 15 min, after which lL was spotted onto H2O-prewashed polyethyleneimine cellulose TLC plates (Macherey & Nagel, 1316 Duren, Germany) that were developed in AcOH : m ă NH4Cl : H2O, : 10 : 85 The stability of the aminoacyltRNA link under the acidic conditions of nuclease treatment was confirmed by separate experiments Radioactivity was detected by phosphorimager analysis (PharosFX Plus; Bio-Rad, Munich, Germany), and quantified using quantityone software (Bio-Rad) Kinetic constants were ´ calculated using sigmaplot (Systat, San Jose, CA, USA) Acknowledgements This work was supported in part by the Deutsche Forschungsgemeinschaft 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Igloi and E Schiefermayr Arginyl-tRNA synthetase amino acid discrimination Table Quantification of discrimination between L-arginine and L-canavanine using jack bean transcript tRNA Assays were based... E.coli transcript Fig Comparison of canavanine incorporation The amount of L-canavanine transferred by arginyl-tRNA synthetase from E coli (waved shading), jack bean (vertical dashes) and soybean (diagonal