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

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 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
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 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

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 tRNA
Arg
ACG
gene from
Canavalia ensiformis established its identity to the Ara-
bidopsis sequence (accession number NR_023294). The

NH OH
L-Arginine
H
2
NN
NH
2
NH
2
NH
2
NH
2
O
OH
O
L-Canavanine
Fig. 1. Structures of L-arginine and its guanidinooxy analogue,
L-canavanine.
Arginyl-tRNA synthetase amino acid discrimination G. L. Igloi and E. Schiefermayr
1308 FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS
tRNA as measured by aminoacylation (data not
shown).
Using either [
14
C]-canavanine in the conventional
aminoacylation assay, or unlabelled canavanine
together with [
32
P]-labelled jack bean transcript tRNA,

Fig. 3. Dependence of the pyrophosphate exchange reaction on
tRNA. The pyrophosphate exchange reaction was carried out in the
absence (
) or the presence of 3 lM ( )or30lM (r) transcript tRNA
or 12 l
M (d) periodate-oxidized jack bean transcript tRNA using jack
bean arginyl-tRNA synthetase. PPi, tetrasodium pyrophosphate.
G. L. Igloi and E. Schiefermayr Arginyl-tRNA synthetase amino acid discrimination
FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1309
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 (K
M
for soybean
56 lm; K
M
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 para-
meters correspond to a discrimination factor, (k
cat
⁄
K
M

cat
⁄ K
M
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
canavanine-producing plant. An additional classic
post-transfer proofreading mechanism [7,29] would
require the rapid deacylation of Cav-tRNA
Arg
by the
L
A -
g r
i
n
i
n
e
L
H
-
o
m
o
a
g r
i

m
o c
i
t
r
u
l
l
i
n e
L
h T -
i
o
c
i
t
r
u
l
l
i
n
e
L
L -
y
s
i
n

100
120
NH
NH
N H
2
NH
2
OH
O
NH
O
NH
N H
2
NH
2
OH
NH
O
S
N H
2
NH
2
OH
NH
2
O
NH

O
OH
NH
O
NH
2
N
N H
2
NH
2
O
OH
O
c a o n i m A y n o i t a l
(
r a % g n i y n o i t a l
)

tRNA
Origin
Aminoacyl-A
76
n a e b k c a J
n a e b y o S
g r A
v a
C
g
r

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
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 tRNA
Arg
ACG
(Fig. 7) despite the fact that
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 [
14
C]-labelled amino acids or [
32

cat
⁄ K
M
(M
)1
Æmin
)1
) K
M
(lM)
k
cat
⁄ K
M
(M
)1
Æmin
)1
) K
M
(lM)
k
cat
⁄ K
M
(M
)1
Æmin
)1
) K

60
80
100
120
Fig. 5. Stability of canavanyl-tRNA. Jack bean transcript tRNA
Arg
that had been aminoacylated with [
14
C]-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, [
14
C]-L-arginyl-tRNA was incubated in the absence of
enzyme ()).
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Jack bean
transcript
Soybean
transcript
Wheat

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
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
reasonable basis for assuming an evolution of a
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 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

Aminoacylation
(pmol ArgÆpmol
)1
tRNA) K
M
(lM)
Aminoacylation
(pmol ArgÆpmol
)1
tRNA) K
M
(lM)
Aminoacylation
(pmol ArgÆpmol
)1
tRNA) K
M
(lM)
Aminoacylation
(pmol ArgÆpmol
)1
tRNA) K
M
(lM)
E. coli 0.64 ND 0.07 ND 0.1 ND 0.11 ND
Jack bean 0.61 0.86 0.05 1.2 0.06 ND 0.09 ND
Soybean 0.46 1.5 0.06 1.1 0.05 ND 0.11 ND
0
10
20

14
C]-L-canavanine relative to the corresponding arginine
incorporation.
Arginyl-tRNA synthetase amino acid discrimination G. L. Igloi and E. Schiefermayr
1312 FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 pK
a
of the guanidine group, lowering the
value from 12.5 by more than 5 pK
a
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 the

values result
in discrimination factors of approximately 40 and 485
for the respective enzymes.
However, in a heterologous system using either
native E. coli tRNA
Arg
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
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 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-

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
tRNA
Arg
in vitro and incorporation of this allelochemi-
cal into proteins in vivo [30,43]. However, the several
hundred-fold discrimination measured for the jack
G. L. Igloi and E. Schiefermayr Arginyl-tRNA synthetase amino acid discrimination
FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS 1313
bean enzyme is considerably lower than the factor of
10
4
normally expected from systems that rely on an
active proofreading process to correct misrecognized
substrates [8]. Nevertheless, physiological evidence
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

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
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
(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-
Planck-Institute, Go
¨
ttingen), while that from E. coli in
recombinant form was provided by A. Weiner (University of
Washington School of Medicine, Seattle, WA, USA). [
14
C]-
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

comparison ( />Gene for arginyl-tRNA synthetase
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
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 T
17
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).
Arginyl-tRNA synthetase amino acid discrimination G. L. Igloi and E. Schiefermayr
1314 FEBS Journal 276 (2009) 1307–1318 ª 2009 The Authors Journal compilation ª 2009 FEBS
Gene for tRNA
Arg
ACG
from jack bean
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-

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
tRNA
Arg
ACG
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 tRNA
Arg
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
performed in T7 RNA polymerase buffer (40 mm Tris ⁄ HCl
pH 8, 12 mm MgCl
2

C]-
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 ° C.
Synthesis of [
14
C]-L-canavanine
[Guanidino-
14
C]-l-canavanine was synthesized essentially as
described previously [60] from 46 lmol canaline free base
and 2 mCi barium [
14
C]-cyanamide (57.5 mCiÆmmol
)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
G. L. Igloi and E. Schiefermayr Arginyl-tRNA synthetase amino acid discrimination

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
MgCl
2
,4mm ATP and the appropriate amount of [
14
C]-
amino acid, tRNA and arginyl-tRNA synthetase. Amino
acid incorporation was followed using 3 MM filter discs
(Whatman, Dassel, Germany) that had been pretreated with
50 lL 5% trichloroacetic acid (to reduce non-specific back-
ground, particularly when using [
14
C]-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 min 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 [
32
P]-labelled tRNA was used. The tRNA

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 (Ig9 ⁄ 4). We thank Dr Gerald
Rosenthal for advice on the synthesis of [
14
C]-l-cana-
vanine.
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