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, Scha¨ nzlestr. 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 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.
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
Abbreviations L-Cav, L-canavanine; PCAF, pentacyanoamidoferroate.
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Results
the participation of
[l-2-amino-4-(guanidinooxy)butyric acid],
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 trans- formed 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 mgÆL)1 cell culture) compares with 1.2 mgÆL)1 cul- ture 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 synthe- tase 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 exten- sion compared to the native enzyme.
Sequence analysis of the tRNAArg
resembles an induced fit type of substrate selection, including 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 biosyn- thesis has, in the past, been largely devoted to prok- aryotes and lower eukaryotes (yeast). With isolated exceptions in the early literature, aminoacyl-tRNA synthetases from plants, which must not only discrimi- nate between the 20 common amino acids but must also contend with related potentially toxic natural ana- logues [15,16], have been ignored. This challenge faced by plants offers a natural alternative to targeted muta- genesis 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 vari- ants, 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] 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 argi- nyl-tRNA synthetases from a canavanine producer (jack bean, Canavalia ensiformis) and from a related (soybean, Glycine max) provides an non-producer opportunity to gain insight into the mechanism of amino acid recognition in the arginine system.
NH2
O
HN
NH
OH
NH2
L-Arginine
ACG gene from Canavalia ensiformis established its identity to the Ara- bidopsis sequence (accession number NR_023294). The subsequent appearance in the NCBI trace archives of a soybean corresponding to the gene of sequence tRNAACG (accession number gnl|ti|1583039205) con- firmed its similarity to the jack bean sequence with a single base difference from A (jackbean) to G (soy- bean) at position 37. The chemically synthesized gene for tRNAArg ACG from jack bean was cloned, and the full-length tRNA was generated by in vitro transcrip- tion. The transcript could be aminoacylated with argi- nine to a level of approximately 0.05 pmol amino acid ⁄ pmol tRNA. The corresponding soybean tran- script had an arginine acceptance level of approxi- mately 0.1 pmol amino acid ⁄ pmol tRNA.
O
NH2
O
N
OH
H2N
NH2
L-Canavanine
stimulate pyrophosphate
Fig. 1. Structures of L-arginine and its guanidinooxy analogue, L-canavanine.
As is the case for arginyl-tRNA synthetases from the pyrophosphate exchange other sources [24–26], reaction is absolutely dependent on the presence of aminoacylatable tRNA. Periodate-oxidized tRNA, which has been shown to be inactive in aminoacyla- exchange tion, did not (Fig. 3). The tRNA concentration dependence of this reaction gives a KM value that is equivalent to that of
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Fig. 2. 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.
120
tRNA as measured by aminoacylation (data not shown).
100
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(
80
assay,
unlabelled
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0
P P
–20
0
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4
6
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12
14
16
Time (min)
and l-albizziine
) or the presence of 3 lM (
Fig. 3. Dependence of the pyrophosphate exchange reaction on tRNA. The pyrophosphate exchange reaction was carried out in the ) or 30 lM (r) transcript tRNA absence ( or 12 lM (d) periodate-oxidized jack bean transcript tRNA using jack bean arginyl-tRNA synthetase. PPi, tetrasodium pyrophosphate.
Using either [14C]-canavanine in the conventional canavanine or aminoacylation 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 argi- nyl-tRNA synthetases from the two plants show differ- ent specificities towards other arginine analogues, the [32P]-labelled tRNA assay was used to screen a selec- tion of amino acids, including ones that have previ- ously been shown not to be substrates for the enzyme from other sources. l-thiocitrulline and the naturally occurring l-homoarginine, l-citrulline, l-homocitrul- (l-2-amino-3-ureidopropanoic line acid) were, at 1 mm concentration, if at all, extremely poor substrates for both plant enzymes (Fig. 4), and
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g r A
v a C
g r A
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Aminoacyl-A76
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Soybean enzyme
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Jack bean enzyme
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NH 2
O
NH
O
OH
NH 2
OH
N H 2
NH 2
OH
O
NH
O
OH
NH2
NH
S
O
NH 2
O
O
N H 2
NH
NH
NH 2
NH
S
NH2 O
NH
O
NH 2
NH2
N
N H 2
N H 2
NH
NH
NH 2
N H 2
O
NH 2
Fig. 4. Quantitative comparison of amino acid utilization by the plant arginyl-tRNA synthetases. The aminoacylation level attained in the pres- ence of L-arginine was compared to that in the presence of 1 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 char- acterized 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 trans- ferred 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 tRNA transcript by the arginyl-tRNA the plant synthetase from soybean. In this case, the kinetic para- meters 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 cana- vanine using the [32P]-labelled tRNA assay [28]. For the jack bean enzyme, a distinct discrimination between
arginine and canavanine for aminoacylation of the plant tRNA transcript was observed when using [14C]-canava- nine. 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 concen- trations 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 fac- tor 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 classic canavanine-producing plant. An additional post-transfer proofreading mechanism [7,29] would require the rapid deacylation of Cav-tRNAArg by the
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Table 1. 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 [32P]-labelled transcript tRNA
Aminoacylation of transcript tRNA with [14C]-labelled amino acid
Arg
Cav
Arg
Cav
kcat ⁄ KM (M
kcat ⁄ KM (M
kcat ⁄ KM (M
kcat ⁄ KM (M
Source of enzyme
)1Æmin)1)
)1Æmin)1)
)1Æmin)1)
)1Æmin)1)
KM (lM)
KM (lM)
KM (lM)
KM (lM)
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 corre- spond to a half life of only approximately 5 min for Cav-tRNAArg even in the absence of either enzyme. In contrast, the half life of Arg-tRNAArg is 46 min. Addition of arginyl-tRNA synthetase from jack bean does not further decrease the stability of the canavany- lated species.
that
The role of tRNA as a cofactor for aminoacylation in those aminoacyl-tRNA synthetases 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 speci- fied. 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, tran- scripts 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 that
tRNAArg
ACG (Fig. 7) despite the fact
100
)
%
140.00
80
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120.00
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100.00
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A N R
40
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60.00
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i
40.00
t - l y c a o n i m A
0
20.00
w d e n a t t a %
(
0.00
0
5
10
25
30
35
20 15 Time (min)
E.coli transcript
Jack bean transcript
Soybean transcript
E.coli native tRNA-Arg
Bovine liver total tRNA
Wheat germ total tRNA
Source of tRNA
Fig. 6. 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.
Fig. 5. 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 ()).
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Table 2. 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 transcript
Jack bean transcript
Soybean transcript
E. coli native tRNAArg
Source of enzyme
Aminoacylation (pmol ArgÆpmol)1 tRNA)
Aminoacylation (pmol ArgÆpmol)1 tRNA)
Aminoacylation (pmol ArgÆpmol)1 tRNA)
Aminoacylation (pmol ArgÆpmol)1 tRNA)
KM (lM)
KM (lM)
KM (lM)
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
tRNA,
E. coli tRNA is a good substrate for arginylation. The presence of E. coli 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 canav- anylation 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
Canavalia ensiformis [17] were reported. However, the pyrophosphate exchange assay, in the absence of the absolutely required tRNA [24], was used to study sub- strate specificity. The apparent arginine activation described may be due to a co-purified lysyl-tRNA syn- thetase (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 for assuming an evolution of a reasonable basis discriminating function by the jack bean enzyme, we considered that investigation of a natural, discriminat- ing ⁄ non-discriminating pair of enzymes would provide further insight into this process.
The evidence that the arginyl-tRNA synthetase of a canavanine producer, e.g. jack bean (Canavalia ensifor- mis), can discriminate between l-arginine and its ana- logue is indirect. It relies on the observation that jack bean plants injected with radioactive l-canavanine do not incorporate the label into their proteins, compared to soybean plants, which do [30]. In a previous study, ‘somewhat indefinite’ conclusions regarding activation of canavanine by the arginyl-tRNA synthetase from
100
The translated gene sequences proved to be 85% iden- tical to each other but had only 25% identity to the yeast enzyme, the only eukaryotic arginyl-tRNA synthe- tase whose 3D structure has been elucidated to date [33]. Despite this limited similarity and the fact that arginyl- tRNA 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.
90
80
70
) n o i t a t r o p r o c n
i
60
50
g r A %
(
40
n o
i t
l
30
20
a y n a v a n a C
10
0
Jack bean transcript
Soybean transcript
Wheat germ total tRNA
Bovine liver total tRNA
E.coli native tRNA-Arg
E.coli transcript
Fig. 7. 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 to the tRNA species indicated was quantified using shading) 0.4 mM [14C]-L-canavanine relative to the corresponding arginine incorporation.
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 (soy- bean)]. 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) do 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
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Arginyl-tRNA synthetase amino acid discrimination
reveals a discriminatory ability that has characteristics approaching those of the jack bean enzyme.
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-carbox- ylate, 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 pres- ence of the oxygen atom in canavanine dramatically influences the pKa of the guanidine group, lowering the value from 12.5 by more than 5 pKa units [36,37], lock- ing the molecule in an imino-oxy tautomer (Fig. 1) and resulting in a largely uncharged side chain at physiolog- ical pH.
Transcripts derived from the sequences of
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 misacyl- ated tRNA. For arginyl-tRNA synthetase, rearrange- ment of the enzyme active site appears to rely on additional discriminatory elements within the tRNA structure to ensure accurate formation of aminoacyl- tRNA. This is reminiscent of the glutamyl- and gluta- minyl-tRNA synthetases of E. coli. For glutamyl-tRNA synthetase, the presence of tRNA eliminates non-speci- fic 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 ele- ments 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 discrimina- tion. 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
the tRNAArg ACG 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 sys- tem [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 the soybean enzyme effectively tRNA. In contrast, 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,
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 hydroly- sed at neutral pH even in the absence of added enzyme. This instability (half life of approximately 5 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 degra- dation. 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 allelochemi- cal into proteins in vivo [30,43]. However, the several hundred-fold discrimination measured for the jack
in a heterologous system using either native E. coli tRNAArg ICG or a transcript of the corre- sponding gene, we observed how the structure of the tRNA itself can modulate the efficiency of discrimina- tion. Whereas these tRNAs are arginylated efficiently by the synthetases from E. coli, jack bean and soy- bean, and although canavanylation to a high level is achieved by the E. coli enzyme, the soybean enzyme
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from E. coli
to what extent
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 evidence [8]. Nevertheless, physiological indicates that canavanine producers do not incorporate this toxic analogue into their proteins. A discrimina- tion 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, 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.
(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 Max- in Planck-Institute, Go¨ ttingen), while that recombinant form was provided by A. Weiner (University of Washington School of Medicine, Seattle, WA, USA). [14C]- l-arginine (12.8 GBqÆmmol)1) was purchased from Perkin- Elmer (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-isothio- ureidovaleric 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 pur- chased from Sigma. E. coli tRNA enriched in tRNAArg ACG to an arginine acceptance of 760 pmol ⁄ A260 was obtained from an expression construct provided by G. Eriani (Institut de Biologie Mole´ culaire 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 3 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 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-orni- thine, 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 pro- teins [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 tauto- meric form. As we have shown that l-thioarginine is an effective and equally good substrate for the arginyl- tRNA 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
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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 The gene for the enzyme from jack bean has been charac- terized recently [23] (accession number AM950325). For the soybean sequence (accession number FM209045), the translated cDNA sequence of Arabidopsis arginyl-tRNA (accession numbers NM_118763 and NM_ synthetase 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 arginyl- tRNA synthetase was used to generate primers for stepwise 5¢ RACE elongation of the sequence [55]. PCR products were purified using Montage cartridges (Millipore, Esch- born, Germany).
G. L. Igloi and E. Schiefermayr
Arginyl-tRNA synthetase amino acid discrimination
Gene for tRNAArg
ACG from jack bean
performed in T7 RNA polymerase buffer (40 mm Tris ⁄ HCl pH 8, 12 mm MgCl2, 5 mm dithiothreitol, 1 mm spermidine HCl, 4% polyethylene glycol 8000, 0.002% Triton X-100), 5 mm NTP, 20 mm GMP, 0.1 units of inorganic pyrophos- phatase (Sigma), 0.7 nmol template DNA, and 52 nm T7 RNA polymerase prepared from the recombinant pAR1219 expression plasmid [58]. Incubation was performed for 4 h at 37 (cid:2)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 (cid:2)C in water, fol- lowed by slow cooling in the presence of 25 mm Tris-HCl, pH 7.5, 250 mm NaCl, 5 mm MgCl2.
Colorimetric detection of canavanine
(ICN Biomedicals, Aurora, OH, USA)
ACG from Arabidopsis
tRNAArg Canavanine detection and quantification were achieved by following its colour reaction with pentacyanoamidoferroate (PCAF) [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, 2 lL 1% potassium persulphate and 5 lL 1% PCAF in water. The colour was allowed to develop for 40 min at room temperature and the absorbance at 530 nm was measured.
Preparation of L-canaline
Total tRNA from jack bean was obtained from cellular RNA by extraction with 1 m NaCl, and purified by DEAE- Sephadex chromatography as described previously [56]. tRNA (1 lg) was ligated to 20 pmol of a 5¢-phosphory- lated, 3¢-periodate-oxidized hybrid RNA ⁄ DNA oligonucleo- tide [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 tran- scription (binding region in lower case), and prevent self- ligation 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, 1 lL of the ligation product was annealed to 1 pmol of universal M13 primer, and the reaction was performed under stan- dard conditions using 15 units of Thermoscript reverse transcriptase (Invitrogen). After incubation for 1 h at 56 (cid:2)C, the reaction was terminated by heating to 85 (cid:2)C for 5 min, followed by RNase H treatment (GE Healthcare) for 20 min at 37 (cid:2)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¢ (accession terminus of 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 tRNAArg ACG sequences available in the databases possess a G:U base pair in the acceptor stem (data not shown).
Protein expression
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 Canava- lia brasiliensis. The extract enriched in arginase was used immediately for preparative-scale conversion of canavanine to canaline. Canaline was recovered from the reaction mix- ture 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 desic- cated at )20 (cid:2)C.
Synthesis of [14C]-L-canavanine
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 followed by an 80 mm on Source15Q (GE Healthcare) 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
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[Guanidino-14C]-l-canavanine was synthesized essentially as described previously [60] from 46 lmol canaline free base (57.5 mCiÆmmol)1, and 2 mCi barium [14C]-cyanamide 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 The genes for jack bean and soybean tRNAArg ACG 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
G. L. Igloi and E. Schiefermayr
Arginyl-tRNA synthetase amino acid discrimination
silica (EtOH : AcOH : H2O, 65 : 1 : 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 mCiÆmmol)1). The stock solution was stored at )70 (cid:2)C in the presence of 2% ethanol.
that were developed in AcOH : 1 m Du¨ ren, Germany) NH4Cl : H2O, 5 : 10 : 85. The stability of the aminoacyl- tRNA link under the acidic conditions of nuclease treat- ment was confirmed by separate experiments. Radioactivity (PharosFX was detected by phosphorimager analysis Plus; Bio-Rad, Munich, Germany), and quantified using quantityone software (Bio-Rad). Kinetic constants were calculated using sigmaplot (Systat, San Jose´ , CA, USA).
Pyrophosphate exchange
Acknowledgements
This work was supported in part by the Deutsche Forschungsgemeinschaft (Ig9 ⁄ 4). We thank Dr Gerald Rosenthal for advice on the synthesis of [14C]-l-cana- vanine.
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