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Acta Biochim Biophys
Sin 2008, 40: 539-553
doi:10.1111/j.1745-7270.2008.00435.x
Amino acid modifications on
tRNA
Jing Yuan1#, Kelly Sheppard1#, and Dieter S?ll1,2*
1Department of Molecular Biophysics and
Biochemistry and 2Department of Chemistry, Yale University, New Haven, CT
06520-8114, USA
Received: March 26,
2008
Accepted: April 15,
2008
This work was
supported by grants from the Department of Energy (DE-FGO2-98ER20311), the
National Institute of General Medical Sciences (GM22854) and the National
Science Foundation (MCB-0645283)
#
These
authors contributed equally to this work
*Corresponding
author: Tel, 1-203-432-6204; Fax, 1-203-432-6202; E-mail, [email protected]
The accurate
formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the
fidelity of translation. Most amino acids are esterified onto their cognate
tRNA isoacceptors directly by aa-tRNA synthetases. However, in the case of four
amino acids (Gln, Asn, Cys and Sec), aminoacyl-tRNAs are made through indirect
pathways in many organisms across all three domains of life. The process begins
with the charging of noncognate amino acids to tRNAs by a specialized
synthetase in the case of Cys-tRNACys formation or
by synthetases with relaxed specificity, such as the non-discriminating
glutamyl-tRNA, non-discriminating aspartyl-tRNA and seryl-tRNA synthetases. The
resulting misacylated tRNAs are then converted to cognate pairs through
transformation of the amino acids on the tRNA, which is catalyzed by a group of
tRNA-dependent modifying enzymes, such as tRNA-dependent amidotransferases,
Sep-tRNA:Cys-tRNA synthase, O-phosphoseryl-tRNA kinase and
Sep-tRNA:Sec-tRNA synthase. The majority of these indirect pathways are widely
spread in all domains of life and thought to be part of the evolutionary process.
Keywords aminoacyl-tRNA; indirect pathways;
tRNA-dependent amidotransferase; tRNA-dependent cysteine biosynthesis;
selenocysteine biosynthesis
In translation, aminoacyl-transfer RNAs (aa-tRNAs) are employed to
convert genetic information stored in messenger RNA sequences to the
three-dimensional information manifested in the resulting proteins.
Aminoacyl-tRNA synthetases (aaRSs) play a crucial role in maintaining the
fidelity of translation by matching each standard amino acid found in proteins
to the corresponding tRNA isoacceptors and forming a cognate aa-tRNA pair. The
aminoacylation reaction is carried out as a two-step process [1]:1 ATP+aa+aaRS ®
aaRS:aa~AMP+PPi2 tRNA+aaRS:aa~AMP ®
aaRS+aa-tRNA+AMPThe first step is the activation of an amino acid with ATP. The
aaRSs produce an aminoacyl adenylate by attaching the carboxyl group in the
amino acid to the phosphoryl group of AMP. In the second step, the activated
amino acid is transferred to the 2 or 3 hydroxyl group of the 3 terminal ribose
moiety of tRNA and followed by release of the final product, aa-tRNA. In the
classical view, 20 aaRSs catalyze the formation of 20 different aa-tRNA pairs.
Each synthetase specifically recognizes a set of tRNA isoacceptors and charges
them with the correct amino acid that corresponds to the anticodons of the tRNA
molecules. The first exception to this one synthetase/one set of tRNAs/one
amino acid rule was discovered 40 years ago [2], when it was shown that Bacillus
Gln-tRNAGln is synthesized from Glu-tRNAGln rather
than from direct acceptance of Gln on tRNAGln.
Thirty years later, the nature of the enzymes catalyzing such tRNA-dependent
amino acid transformations was uncovered [3]. With advances in functional
genomics as well as in biochemical and genetic analyses, the indirect pathways
for Gln-tRNAGln, Asn-tRNAAsn, Cys-tRNACys and
Sec-tRNASec formation have been characterized [4–7]. They all require two
types of enzymes: aaRSs, which can form misacylated intermediates, and
tRNA-dependent amino acid-modifying enzymes, which convert tRNA-bound amino
acid to form the cognate aa-tRNA pair. Organisms that posses one or more of
these indirect pathways do not have to encode the full set of 20 aaRSs [5,8–13], once
thought to be essential for all living species. The occurrence of tRNA-dependent amino acid transformations is
surprisingly widespread throughout all three domains of life (Table 1).
All known archaea [5], most bacteria [8], and chloroplasts [14,15] encode the
indirect pathway for Gln-tRNAGln formation. A A
non-discriminating GluRS (ND-GluRS) aminoacylates tRNAGln with
Glu [16,17]. A Glu-tRNAGln amidotransferase (Glu-AdT)
then recognizes the mischarged species and transforms it to Gln-tRNAGln in the presence of ATP and an amide donor [Fig. 1(A)] [5].
Likewise, for Asn formation, Asp is first ligated to tRNAAsn by a ND-AspRS [17,18] and then converted to Asn on the tRNA by an
Asp-tRNAAsn amidotransferase (Asp-AdT) [Fig. 1(B)] [18,19]. The
ND-AspRS/Asp-AdT pathway is present in most bacteria and archaea [5,8,9]. In a
large subset of euryarchaeota, a Cys-tRNACys is
formed via an O-phosphoseryl-tRNACys
(Sep-tRNACys) intermediate catalyzed by a noncanonical synthetase, SepRS. The
tRNA bound O-phosphoserine (Sep) is then converted to Cys by SepCysS in
the presence of a sulfur donor (Fig. 2) [6]. Selenocysteine formation
pathways are exclusively tRNA-dependent and found in all Sec-decoding organisms
[13]. In archaea and eukaryotes, it is a multistep process involving SerRS, PSTK
and SepSecS (Fig. 3) [7,20]. Ser is esterified onto tRNASec by SerRS and then phosphorylated by PSTK on the tRNA forming Sep,
which is further converted to Sec by SepSecS. The archaeal and eukaryotic
pathway is different from the bacterial one, where the mischarged Ser-tRNASec is directly converted to Sec-tRNASec by
selenocysteine synthase (SelA) [13]. In this review, we summarize the latest
progress in characterizing the enzymes involved in tRNA-dependent amino acid
transformations and the current evolutionary views of these pathways.
Bacterial tRNA-dependent
Amidotransferase and tRNA Recognition
In bacteria, the tRNA-dependent amidation of Glu and Asp to form
Gln-tRNAGln and Asn-tRNAAsn is catalyzed by the same
enzyme: the heterotrimeric GatCAB [3]. The exact functional role of bacterial
GatCAB in vivo is determined by the availability of its misacylated
substrates Glu-tRNAGln and Asp-tRNAAsn
[3,4,21]. In bacteria that only have a ND-GluRS (e.g. Bacillus subtilis)
[16], Glu-tRNAGln is generated and GatCAB is exclusively used
as a Glu-AdT to transform Glu-tRNAGln to Gln-tRNAGln [3]. In bacteria that only encode a ND-AspRS [e.g. Deinococcus
radiodurans, Neisseria meningitides, Pseudomonas aeruginosa and
Thermus thermophilus (T. thermophilus)], GatCAB functions as an
Asp-AdT, synthesizing Asn on tRNAAsn [4,18,21–26]. However, in
bacteria possessing both a ND-GluRS and a ND-AspRS (e.g. Chlamydia trachomatis
[27] and Helicobacter pylori [28–30]), GatCAB catalyzes both
tRNA-dependent transamidation reactions in vivo [8,27,31]. In general, the anticodon regions of the tRNAGln and the tRNAAsn, respectively, are not the
major identifying elements for recognition by tRNA-dependent amidotransferases
[26,32–34]. In order to discriminate against tRNAAsp and
tRNAGlu, bacterial GatCAB recognizes the first base pair in the tRNA
acceptor stem, which is U1-A72 in tRNAGln [32] and tRNAAsn [26] and G1-C72 in tRNAAsp and tRNAGlu. Additionally, the supernumerary nucleotide U20a in the D-loop of tRNAAsp and tRNAGlu excludes them as substrates for GatCAB [32]. In N. meningitides,
the mutation of U1-A72 to G1-C72 in tRNAAsn leads
to a 100-fold decrease in transamidation activity, while the transplantation of
the U1-A72 into tRNAAsp, together with the deletion of the U20a, converts tRNAAsp into a substrate that behaves
similarly to the wild-type tRNAAsn for bacterial GatCAB [26].
Sequence comparisons of bacterial tRNAGlu, tRNAGln, tRNAAsp and tRNAAsn have
shown that the first base pair of tRNAAsn and tRNAGln is conserved as U1-A72 in all bacteria encoding tRNA-dependent
pathways for Gln and Asn biosynthesis, suggesting that GatCAB utilizes one
general mechanism to maintain its substrate specificity [26,32].
Archaeal tRNA-dependent
amidotransferases and tRNA recognition
Two types of tRNA-dependent amidotransferases (AdTs) are found in
archaea: the heterodimeric GatDE and the archaeal GatCAB [5]. GatDE is the
archaeal Glu-AdT [5]. It exclusively recognizes archaeal tRNAGln and synthesizes glutamine on the tRNA. The archaeal GatCAB only
exists in archaea lacking an asparaginyl-tRNA synthetase (AsnRS) [5,9]. In
vitro the Methanothermobacter thermautotrophicus (M.
thermautotrophicus) GatCAB only acts on Asp-tRNAAsn but
not on homologous Glu-tRNAGln, suggesting that the archaeal
GatCAB has lost its dual function and acts strictly as an Asp-AdT [35]. The
advantage of a more specialized archaeal GatCAB compared to its bacterial
homolog awaits further investigation.The tRNA recognition mode of archaeal GatCAB diverges from the
bacterial enzyme. The archaeal GatCAB does not recognize the first base pair of
the tRNA substrate; instead, in the case of M. thermautotrophicus
GatCAB, it discriminates against tRNAAsp by recognizing U49 and
the D-loop as major anti-determinant elements [33]. A mutation of A49 to U49 in
the wild-type tRNAAsn leads to loss of more than 99.5% of activity
in the archaeal GatCAB catalyzed transamidation reaction. Additionally, the
length of the variable loop acts as an identity element in tRNAAsn recognition as demonstrated by the M. thermautotrophicus and
Methanosarcina barkeri enzymes [26,33]. While the recognition of tRNAGln by GatDE, the archaeal Glu-AdT, relies on the same regions (i.e. the
first base pair and the D-loop) as the bacterial GatCAB, the bases recognized
are different. GatDE recognizes the first base pair of archaeal tRNAGln, conserved as A1-U72, whereas bacterial GatCAB recognizes the
U1-A72 base pair of bacterial tRNAGln or tRNAAsn [34]. Mutation of U19 or A20 in the D-loop of M.
thermautotrophicus tRNAGln significantly decreases
transamidation catalyzed by GatDE [34].
The catalytic mechanism of
tRNA-dependent amidotransferases
The catalytic mechanism of
tRNA-dependent amidotransferases
Transamidation is an ATP-dependent, multistep reaction requiring the
presence of an amide donor such as glutamine or asparagine. Despite the
difference in tRNA specificity and natural distribution, GatCAB and GatDE use
the same mechanism to catalyze tRNA-dependent transamidation (Fig. 4).
It consists of three sub-reactions: (a) the activation of the amide acceptor
(tRNA-bound Glu or Asp) at the expense of ATP hydrolysis, forming g-phosphoryl-Glu-tRNAGln or possibly b-phophoryl-Asp-tRNAAsn as reaction intermediates
[15,36–38]; (b) the hydrolysis of an amide donor Gln or Asn to form enzyme
captivated ammonia; and (c) the transfer of sequestered ammonia to the
activated intermediate to form the final product Gln-tRNAGln or Asn-tRNAAsn [38–41]. The kinase (a) and the
glutaminase activity (b) of AdTs are tightly coupled upon binding of the
misacylated tRNA substrate [8,38,40,42].GatDE forms an a2b2
tetramer in solution and crystalline conditions [Fig. 5(A)] [34,42]. The
functional role of each subunit in GatDE is well elucidated. The D subunit
carries out the glutaminase activity [5,38,42], which releases ammonia from Gln
as well as Asn [8,35]. It consists of three domains: AnsA-like domain 1,
AnsA-like domain 2 and the-N-terminal domain. The AnsA-like domains connect
through a long linker loop to the N-terminal domain, which is involved in the
binding of the E subunit [34,42]. The D subunit forms a tightly packed dimer
with a large surface contact area located at the AnsA-like domains between the
two protomers. In the dimer interface, two amidase catalytic centers are formed
by AnsA-like domains 1 and 2 from the other subunit [42], where two highly
conserved threonine residues, an aspartic acid and a lysine, are crucial for
the GatD-catalyzed glutaminase activity [38]. GatD only hydrolyzes Gln in the
presence of the E subunit and Glu-tRNAGln, which couples the
hydrolysis of Gln with the activation of tRNA-bound Glu, thus preventing an
otherwise futile deamination of Gln and the accumulation of free ammonia [38].GatE interacts with the misacylated tRNA substrate [34,38]. In the
presence of ATP, the E subunit alone is able to activate Glu-tRNAGln, forming a g-phosphoryl-Glu-tRNAGln intermediate [38]. Its unique
structure consists of a cradle domain, an AspRS-like insertion domain, a
helical domain and a C-terminal domain homologous to the YqeY protein family,
and it may enhance protein affinity towards its tRNA substrate [34,42]. A
similar C-terminal YqeY domain appended to the D. radiodurans Gln-tRNA
synthetase (GlnRS) enables the enzyme to bind productively to tRNAGln [43]. The helical domain and the C-terminal domain change their
orientation upon tRNA binding and form a concave surface to accommodate the
elbow region of the tRNA substrate [Fig. 5(B)] [34]. The lack of tRNAGln-specific and base-specific interactions in this region of the tRNA
indicates that a shape-complementary mechanism as the indirect readout of tRNAGln is the main factor that allows GatE to differentiate tRNAGln from tRNAGlu and tRNAAsn [34].
The cradle domain interacts with the ACCA-terminus of tRNAGln and guides the attached Glu to the catalytic center for
phosphorylation and the subsequent transamidation reaction [34]. The kinase
activity requires the presence of Mg2+ ions. Mutations of Mg2+ binding residues in the M. thermautotrophicus GatE subunit
drastically affect the enzyme affinity to Mg2+ and
abolish the kinase and transamidase activities in vitro [34].GatCAB and GatDE are evolutionarily related through their kinase
domains (GatB and GatE) [5,44]. The GatB subunit, which contains the catalytic
pocket of the kinase and transamidase activities, is also involved in tRNA
binding [32]. Structurally, bacterial GatB is made of three domains: the cradle
domain, the helical domain and the C-terminal YqeY domain, which has been shown
to participate in tRNA substrate binding [43]. Archaeal GatB is expected to
have different tRNA recognition elements compared to its bacterial homolog, as
the archaeal GatCAB is a strict Asp-AdT. The detailed interaction map between
GatCAB and substrate tRNA is still unknown.The bacterial GatCAB alone has basal glutaminase activity [32,40],
which is enhanced significantly in the presence of its mischarged tRNA
substrate and ATP [8,40]. GatA belongs to the amidase family. Functionally
similar to GatD, it generates active ammonia from an amide donor. The A subunit
is a single domain protein possessing Ser-cis-Ser-Lys as the catalytic
triad. In the Staphylococcus aureus GatCAB crystal structure, a Gln
molecule located in close proximity to the catalytic triad further proves that
subunit A’s function is to liberate ammonia from the amide donor glutamine.
Unlike GatD, bacterial GatA prefers Gln as the amide donor to Asn [8,15,45,46].
The shorter side chain in Asn prevents the b-carboxyl carbon from
interacting with the active site Ser and forming a tetrahedral intermediate,
and thus reduces the deamination efficiency [8,32,46]. In contrast, the
archaeal GatA does not seem to have a preference regarding the amide donor, as
the M. thermautotrophicus enzyme can use either Gln or Asn with almost
equal efficiency [35], which suggests a possible different arrangement of the
active site in archaeal GatA. GatC is a 10 kDa small protein responsible for stabilizing the GatCAB
trimeric protein complex. In the crystal structure of S. aureus GatCAB (Fig.
6), the C subunit is shaped as an extended loop with two helixes at its
N-terminus and two b strands at its C-terminus [32]. GatC stabilizes the GatAB complex
by extensively interacting with both subunits at the GatA/GatB interface [32].
The proper folding of the GatA subunit also requires the presence of GatC [3].
Gated Ammonia Channel in
tRNA-dependent Amidotransferases
A most notable feature in the crystal structures of S. aureus
GatCAB and M. thermautotrophicus GatDE is a long protein tunnel (30 and 40 , respectively) connecting the
glutaminase activity center in the GatA or GatD subunit to the
kinase/transamidase active site in the GatB or GatE subunit [32,34]. The
molecular tunnel is made of continuous hydrophilic residues with highly
conserved positive and negative residues alternating on the inner surface, and
it is surrounded by hydrophobic residues on the outside [32,34]. Ammonia
generated in the deaminase center is expected to travel to the transamidase
center for the transamidation reaction to occur [32,34,42]. The hydrophilic
property of the ammonia tunnel in AdTs suggests that ammonium (NH4+) instead of ammonia (NH3) is transported. It has
been suggested that the transport is carried out through alternating
protonation and deprotonation of the ammonium ion [32]. A continuous
desolvation of ammonium ions, followed by passage into the tunnel, may push
ammonium ions towards the subsequent reaction center, mimicking the mechanism
of K+ transport in the potassium channel [34,47]. The coupling of glutaminase and kinase/transamidase activities has
been observed in both GatCAB and GatDE [8,38,40,42]. Regarding GatDE, only in
the presence of misacylated tRNA substrate does the hydrolysis of Gln or Asn
amide donor occur [38,42]. The binding of the tRNA substrate induces a
significant conformational change in the D subunit where a catalytic threonine,
7 ? away from the active site in the apo enzyme, moves to the active position
[42]. The ammonia tunnel also undergoes conformational changes and it is
proposed to switch from a closed to an open state upon binding of the
misacylated tRNA [32,34,42]. Unlike GatDE, GatCAB has a less tight coupling
between these two subreactions as mentioned earlier [8,32,40]. The lack of a
complex structure of GatCAB and tRNA leaves many questions open in this area.
Complexes between tRNA,
Non-discriminating-aaRS and tRNA-dependent Amidotransferase
Several mechanisms have been proposed to maintain the fidelity of
translation and to prevent the misacylated tRNAs generated during the described
tRNA-dependent amino acid transformations from participating in decoding, such
as EF-Tu discriminating against misacylated tRNAs [48] and substrate channeling
[14,49]. The first mechanism is based on the diverse binding affinity of EF-Tu
towards different tRNA species and their attached amino acids [50]. The cognate
aa-tRNAs bind EF-Tu with similar affinity by thermodynamic compensation,
whereas the matching amino acid of a strong binding tRNA weakly binds to EF-Tu
and vice versa [48]. The tRNAGln and tRNAAsn have weaker affinity toward EF-Tu than tRNAGlu and
tRNAAsp [48]. Therefore, Glu-tRNAGln and Asp-tRNAAsn, as weak/weak combinations, are expected to have considerably less
affinity for EF-Tu than cognate aa-tRNAs and are less likely to be involved in
translation.Some recently reported evidence supports a substrate channeling
mechanism. Structurally, GluRS from T. thermophilus can be docked easily
onto M. thermautotrophicus GatDE:tRNAGln ,
forming a ternary complex [34]. The CCA end of the tRNAGln in the
active center of ND-GluRS can move to the kinase/transamidase center in GatE
via a simple flip motion resembling the movement of tRNA and its aaRS with an
editing domain [34]. A similar complex of ND-AspRS, GatDE and tRNA cannot be
constructed due to large steric clashes. Biochemically, a stable complex of T.
thermophilus ND-AspRS, GatCAB and tRNAAsn has
been observed in vitro and in vivo [51]. The presence of a
ND-aaRS reduces the Km of GatCAB for Asp-tRNAAsn and stabilizes Asp-tRNAAsn as well as the final
product Asn-tRNAAsn [51,52]. Compared to the free enzymes,
complex formation also increases the kcat
of ND-AspRS. Substrate channeling couples aminoacylation with transamidation,
thus increasing the overall reaction efficiency and preventing the
incorporation of misacylated tRNA species in translation.
tRNA-dependent
Amidotransferase in Mitochondria
A number of eukaryotes, including Saccharomyces cerevisiae
[53] and Homo sapiens [44] encode homologs of AdT subunits in their
nuclear genomes. Several lines of evidence suggest that the indirect pathway
for Gln-tRNAGln formation may also be used in the mitochondria of these eukaryotes.
For example, in yeast mitochondria, the activity of Glu-AdT is present and was
first detected nearly three decades ago [54]. Recently, the AdT activity was
also found in mammalian (T. Suzuki, unpublished data) and plant mitochondria
[55]. Furthermore, the yeast AdT homologs (Pet112 and YMR293C) are essential
for mitochondrial function [56,57], and Glu-tRNAGln, the
substrate of Glu-AdT, was found to be located in the mitochondria of S.
cerevisiae [58]. The formation of Glu-tRNAGln is
intriguing, however, as the reaction cannot be catalyzed by the yeast
mitochondrial GluRS in vitro [59]. Additionally, cytoplasmic tRNAGln and GlnRS were shown to be imported to the yeast mitochondria as
well [59]. The import of tRNAGln was also shown for H.
sapiens (J. Alfonzo, unpublished data). It is unclear what may be the
reasons (e.g. additional coding functionality) for the presence of dual
pathways for Gln-tRNAGln formation in mitochondria.
The Evolutionary Ciew of the tRNA-dependent
Gln and Asn Formation Pathways
The indirect pathways for Gln and Asn formation are thought to be
ancient and existed in the last universal communal ancestor (LUCA), while the
corresponding GlnRS and AsnRS in the direct pathways are later additions during
evolution [9,60–63]. The indirect pathway couples amino acid biosynthesis with
translation, and the direct pathway requires a de novo synthesis of Gln
and Asn independent of tRNA. The indirect pathway for the Asn-tRNA formation
can serve as the sole pathway for free Asn formation. In fact, for many
organisms encoding the ND-AspRS/Asp-AdT pathway, the enzymes for Asn
biosynthesis (AsnA and AsnB) are found to be absent suggesting that the
presence of the indirect pathway for Asn formation is essential [8,24]. On the
other hand, organisms possessing AsnA, the ammonia-dependent asparagine
synthetase, Asn-tRNAAsn is always made using AsnRS through the
direct pathway [8,9]. In the case of Gln, bacterial Glu-AdT prefers Gln as the
amide donor [8,15,45,46]thus both direct and indirect pathways rely on the de
novo biosynthesis of Gln. Archaeal Glu-AdT can use both Gln and Asn as the
amide donor [5,35]. Therefore, in archaea possessing AsnA, GatDE could use Asn
as the amide donor for Gln-tRNAGln formation catalyzed by the
Glu-AdT, adding an additional pathway for Gln biosynthesis.The retention of the indirect pathway for Gln formation in Archaea
may be due to the unique archaeal tRNAGln [44], which cannot be
recognized and aminoacylated by GlnRS from Escherichia coli or S.
cerevisiae [5]. On the other hand, bacterial tRNAGln from B.
subtilis, an organism that encodes the indirect pathway, is a good
substrate for E. coli GlnRS [16]. In all free-living organisms, ammonium
is fixed mainly through the conversion of Glu to Gln by glutamine synthetase.
The free amino acid Gln also serves as an amide donor for several other
biosynthetic pathways as well as a signaling molecule for the nitrogen
metabolism [64,65]. A number of bacteria encoding a Glu-AdT have increased
amounts of free Glu in their cells, which favors the indirect pathway for
Gln-tRNAGln formation suggesting another possible reason to maintain the
ancient indirect pathway. The reason why the indirect pathways have not been
replaced by the direct pathways in these bacteria may include nitrogen, carbon
regulation and translation fidelity. The details await further investigation.
tRNA-dependent Cys Synthesis
in Archaea
In a large subset of Euryarchaeota [66], Cys is synthesized
in a tRNA-dependent manner (Table 1) [6]. This indirect pathway for
Cys-tRNACys formation utilizes two enzymes, SepRS and SepCysS (Fig. 2)
[6]. SepRS aminoacylates tRNACys with Sep [6]. The Sep moiety
is then converted to Cys by SepCysS in the presence of an unknown sulfur donor to
form Cys-tRNACys [6].
Genomic analyses revealed
that SepRS and SepCysS are both encoded in the sulfate reducing archaeon Archaeoglobus
fulgidus [67] and all known methanogenic archaea [66] except Methanosphaera
stadtmanae [68] and Methanobrevibacter smithii [69]. In most of
these archaea, CysRS is also coded for though it may not be essential [66,70];
for example, in Methanococcus maripaludis CysRS is dispensable [71].In many of these euryarchaeal genomes the enzymes required for the
formation of free Cys (i.e. tRNA-independent) biosynthesis are not
encoded [6,66]. The indirect route for Cys-tRNACys
formation using SepRS and SepCysS is likely the sole means for Cys biosynthesis
in these organisms [6]. This is consistent with an earlier report demonstrating
that Sep is a precursor for Cys biosynthesis in M. jannaschii [72].
Further studies have shown that an archaeal Sep biosynthetic pathway can
provide sufficient Sep levels for Ser, cystathionine and tRNA-dependent Cys
production [73]. Furthermore, knocking out sepS in M. maripaludis
resulted in a Cys auxotroph [6]. Therefore, the use of SepRS and SepCysS for
the tRNA-dependent Cys synthesis in these organisms likely enables coupling of
protein synthesis with Cys production.
SepRS Directly Aminoacylates
tRNACys
with O-phosphoserine
SepRS is a subclass IIc aaRS like PheRS and PylRS [6,74], sharing a
common ancestor with the a-subunit of PheRS [66,74]. Both biophysical and structural analyses
demonstrate that SepRS is a homotetramer [75,76]. The core of this a4
assembly resembles that of PheRS and consists mostly of the four catalytic
domains [75]. While the active site of SepRS is structurally similar to that of
PheRS, SepRS uniquely recognizes its amino acid substrate, Sep [76]. The
phosphate group of the latter is highly recognized by SepRS with each of the
three non-bridging oxygen atoms forming two hydrogen bonds to residues in the
enzyme’s binding pocket. Mutation of these residues in the M. maripaludis
SepRS resulted in inactive mutant enzymes [75]. The recognition of the
phosphate moiety includes hydrogen bonding between two non-bridging oxygens and
the a-amino group of active site residues, which is unique amongst aaRSs
to SepRS. Structural results suggest that dipole interactions between Sep and a
central a-helix in the active site of SepRS stabilize the polar side chain of
the substrate, another feature not observed in other aaRSs [76].Each monomer of SepRS can recognize and aminoacylate tRNACys in cis [76], in contrast to PheRS where the tRNA anticodon
recognition site and aminoacylation active site are found on different
subunits. However, in the co-crystal structure of the A. fulgidus SepRS
with tRNACys only two tRNA molecules bound to the tetramer [76]. Computer
modeling though does suggest that four tRNAs could be accommodated by the
complex [76]. The stoichiometry in solution of SepRS to tRNACys is not clear and awaits further investigation. Biochemical studies revealed that M. maripaludis SepRS recognizes
the same major identity elements in tRNACys (G34,
C35, and A36) as the homologous CysRS [77]. Both aaRSs also use G15 and A47 in
tRNACys as minor identity elements. However the base pairs G1:C72 and
G10:C25, and nucleotides G37 and A59 serve as minor identity elements for only
SepRS [77]. The use of similar identity elements in tRNACys recognition by both SepRS and CysRS, both of which were present in
LUCA [66,74], suggests that the genetic code predates the modern aminoacylation
machinery [77]. Given that SepRS, like other class II aaRSs, approaches the
tRNA from the major groove side while CysRS, a class I aaRS, approaches it from
the minor groove side has lead to speculation that a complex between SepRS,
tRNACys and CysRS is possible [76], though the in vivo role of such
a complex is currently not clear.
SepCysS Catalyzed Formation of
Cys-tRNACys
The pyridoxal phosphate (PLP)-dependent enzyme SepCysS modifies the
Sep bound to tRNACys to form Cys-tRNACys [6].
The sulfur donor for this enzyme is unknown though in vitro sulfide is
sufficient [6]. The A. fulgidus SepCysS crystal structure (2.4 ?
resolution) [78] revealed that it belongs to the fold type I family [79] with
its large N-terminal domain being comprised of a characteristic seven stranded b-sheet which
typifies this family of enzymes. In addition, the structure showed that the
enzyme forms a homodimer [78]. The active site of the enzyme is formed in a
large basic cleft in the dimer interface and is comprised of conserved residues
from both monomers [78]. Modeling a SepCysS:Sep-tRNACys
complex suggests that a conserved Arg79, His103, and Tyr 104 (A. fulgidus
numbering) recognize the phosphate group of the Sep moiety of the tRNA
substrate [78]. The same work implicates one of the three conserved Cys
residues (39, 42 or 247) in the SepCysS active site as the persulfide sulfur
carrier essential for catalysis (Fig. 7) [78] though this awaits further
study.The crystal structure of SepCysS with PLP alone revealed that the
co-factor formed a Schiff-base linkage with the conserved Lys209 in the active
site of SepCysS [78]. A hydrogen bond between SepCysS and the nitrogen atom of
the ring structure of PLP is achieved through the side chain of a conserved Asn
and not an Asp as is found in most PLP-dependent enzymes [78]. Nevertheless,
like in other PLP-dependent enzymes, the co-factor in SepCysS is thought to
stabilize the negatively charged transition state formed during catalysis of
the b-replacement reaction [80].M. maripaludis encodes both the direct
and indirect paths for Cys-tRNACys synthesis. As noted above, the sole route for Cys
formation is tRNACys dependent. Intriguingly while sepS
(encoding SepRS) can be deleted when the organism is grown in the presence of
Cys, pscS (encoding SepCysS) cannot (T. Major, M. Hohn, D. Su, W.B.
Whitman, unpublished data), raising the question whether SepCysS possesses an
additional function in M. maripaludis that is essential.
Cys Synthesis in Archaea
Four different routes for Cys formation have been discovered in
archaea: the eukaryotic pathway in which the precursor is cystathionine [81],
the bacterial pathway where O-acetylserine serves as the precursor [82–84], a modified
bacterial pathway with free Sep as the precursor [84,85], and the
tRNA-dependent route with Sep-tRNACys as the precursor [6].
Cys is implicated as the major sulfur source for a variety of biosynthetic
pathways including Fe-S cluster formation, tRNA modification, and biosynthesis
of co-factors in bacteria (reviewed in [87]). Fe-S cluster proteins are highly
encoded in the genomes of methanogenic archaea [88]. Whether Cys generated
through the tRNA-dependent pathway is used as the sulfur source is an open area
of investigation. It is thought that these euryarchaea must balance the need
for Cys in protein synthesis and other biosynthetic pathways by controlling the
deacylation of Cys-tRNA, thus regulating the level of free Cys relative to
Cys-tRNACys; however, it may well be that these methanogens which grow in
environments rich in reduced sulfur compounds may be able to use inorganic
sulfur directly.
Evolution of the Two Cys-tRNACys
Biosynthetic Pathways
It has been speculated that the indirect pathways for aa-tRNA
formation predate the direct ones [89]. While phylogenetics supports this
speculation for amide aa-tRNA synthesis (discussed above), analyses using
structure-based amino acid alignments [66,74] suggest that both pathways
(SepRS/SepCysS and CysRS) for Cys-tRNACys formation were present
in LUCA. The phylogenetic data suggest that only early bacteria retained CysRS
while the ancestral archaea possessed SepRS and that CysRS was later
horizontally transferred to archaea. In some archaeal lineages the bacterial
CysRS replaced the indirect pathway for Cys-tRNACys
synthesis while in many euryarchaea CysRS either coexisted with SepRS/SepCysS
or was not retained [66]. Why the indirect pathway for Cys-tRNACys formation has been retained in these euryarchaea remains an open
question. It is speculated that a link
between Cys formation, sulfur metabolism and methanogenesis may exist that
would favor retention of the tRNA-dependent route for Cys biosynthesis [66,90]
though this awaits further experimental inquiry.
tRNA-dependent Sec Formation
Sec is the major biological form of selenium, an essential dietary
trace element in humans implicated in cancer prevention [91,92]. Sec is coded
as the 21st amino acid in a number of species across all three domains of life (reviewed
in [13,93]). Under physiological conditions (pH 7), Sec is more stable in its
ionized form than Cys due to the lower redox potential which thus lowers the pKa of the selenol group of Sec compared
to the thiol group of Cys (5.2 and 8.5, respectively) [94]. Sec thus serves as
an excellent nucleophile in the active sites of proteins involved in
oxidation-reduction reactions.Selenoprotein synthesis requires the formation of
selenocysteinyl-tRNASec (Sec-tRNASec). No
aaRS, [i.e. a selenocysteinyl-tRNA synthetase (SecRS)], has been
identified in Sec-decoding organisms that can carry out the task (Table 1)
and instead Sec is synthesized on tRNASec (Fig. 3). Why
Sec-tRNA is only formed via indirect paths remains unknown but it may be due to
selective pressures to maintain translational fidelity. CysRSs from E. coli
[95] and Phaseolus aureus [96] have the ability to aminoacylate tRNACys with Sec. Therefore, given the fact that misincorporation of Sec in
place of Cys can be detrimental to protein function [97], the levels of free
Sec are likely well regulated and kept low to prevent misacylation of tRNACys with Sec. It is also worth noting that Sec to Cys mutations lead to
mutant enzymes with significantly reduced activities [98]. Synthesizing Sec on
tRNASec enables formation of Sec-tRNASec while
potentially minimizing the free Sec levels in vivo, preventing
misacylation of tRNACys with Sec; thus the retention of the
tRNA-dependent Sec pathway may be a mechanism of ensuring the accurate decoding
of Cys and Sec [99].In all known Sec-decoding organisms [100–102], tRNASec is first serylated by SerRS to form Ser-tRNASec [103–105]. Work in the 1990s revealed in bacteria that selenocyteine
synthase (SelA) transforms Ser bound to tRNASec to Sec
(Fig. 3) [13]. The Sec-tRNASec formed is then used in
protein synthesis to decode UGA (usually a stop codon) when an RNA element, the
selenocysteine insertion sequence element, is present in the mRNA [13]. A
unique elongation factor, SelB, brings the Sec-tRNASec to the
ribosome [13]. The mischarged species, Ser-tRNASec, in
Sec-decoding archaea and eukaryote (Fig. 7) is not directly modified to
Sec-tRNASec, but rather the Ser moiety on the tRNA is phosphorylated by PSTK to
form Sep-tRNASec (Fig. 3) [106,107]. The tRNA-bound
Sep is then converted to Sec by the PLP-dependent enzyme Sep-tRNA:Sec-tRNA
synthase (SepSecS) [7,20]. Like SelA, SepSecS uses selenophosphate as the selenium donor to
produce Sec-tRNASec in vitro and in vivo [7,20,108–110]. SepSecS is
unable to use Ser-tRNASec as a substrate, recognizing
only Sep-tRNASec [7,20]. However, in vitro bacterial
SelA in addition to Ser-tRNASec, can convert Sep-tRNASec to Sec-tRNASec [20], though the biological
relevance of this is not clear as PSTK is not encoded in bacterial genomes [7].
In all known Sec-decoding archaeal and eukaryal genomes, PSTK and SepSecS are
always both encoded [111]. It is unknown why archaea and eukaryotes use Sep-tRNASec as an intermediate in tRNA-dependent Sec biosynthesis. The carboxyl
ester bond between Sep and tRNASec is more stable than Ser and
the tRNASec [106]. In addition, the phosphate moiety of Sep is likely a better
leaving group than the hydroxyl moiety of Ser. Thus, Sep-tRNASec may serve as a better precursor for Sec-tRNASec formation than Ser-tRNASec [7].
tRNASec-dependent Ser Phosphorylation
Work with extracts from rat and rooster liver and lactating bovine
mammary gland in the 1970s first demonstrated that Ser-tRNA could be phosphorylated
[112,113]. While it was later shown with partially purified enzyme from bovine
liver that the enzyme had a high affinity for tRNASec, it
was only in 2004 that the protein (PSTK) was identified from mouse [106], and
soon after the archaeal homolog [107].PSTK phosphorylates Ser-tRNASec by
transferring the g-phosphate of ATP onto the Ser moiety in an Mg2+-dependent manner [106,111]. The enzyme belongs to the P-loop kinase
superfamily [114], possessing a phosphate-binding loop (P-loop), a Walker B
motif, and an RxxxR motif in its N-terminal domain [111]. Mutation of conserved
residues in these motifs in the M. jannaschii PSTK resulted in mutant
enzymes with significantly reduced activity [111]. While PSTK prefers ATP, in
vitro the enzyme is able to use other NTPs (GTP, CTP, UTP and dATP) as
substrates, like T4 polynucleotide kinase [111]. Similar to other members of
the kinase superfamily [114], the ATPase activity of PSTK is activated in the
presence of its other substrate Ser-tRNASec [111].
Interestingly, the activity is also enhanced when unacylated tRNASec is provided [111].
PSTK recognition of Sep-tRNASec
It does not appear that PSTK uses the Sep moiety of tRNASec as a major recognition element as the enzyme has a similar Kd for unacylated tRNASec as Ser-tRNASec (39 nM and 53 nM, respectively) [111]. In vivo, the
concentration of tRNASec is approximately 10% of that of tRNASer [115,116], the other tRNA substrate of SerRS. It may well be that
PSTK serves as a tRNASec scavenger for SerRS [111]. PSTK may also
assist in maintaining translation fidelity by preventing the misacylated tRNASec intermediates from being used in protein synthesis and channeling
Sep-tRNASec to SepSecS [111].Surprisingly, it appears that archaeal and eukaryotic PSTK enzymes
recognize different elements in Ser-tRNASec. While
tRNASec possesses an extended variable loop like tRNASer, a major identity element for SerRS recognition of tRNA [117], it
is distinct from other tRNA isoacceptors by possessing an elongated acceptor stem
and D-stem [118,119]. For tRNASec recognition by eukaryotic
PSTK, the major element is the length and conformation of the elongated D-stem
[120]. For archaeal PSTK, the D-stem is a minor identity element and the G2-C71
and C3-G70 base pairs in the acceptor stem of archaeal tRNASec serve as the major recognition elements in the tRNA [121]. Given
the deep phylogenetic divide between archaeal and eukaryotic PSTK [111], this
may be a strong indication of co-evolution of PSTK and tRNASec [121]. Interestingly, while bacterial tRNASec has an
8 bp acceptor stem and a 5 bp T-stem, and archaeal and eukaryotic tRNASec have a 9 bp acceptor stem and 4 bp T-stem arrangement [118,122–124], PSTK and
SepSecS can use E. coli tRNASec in vivo [7]. In
turn, E. coli can use the human tRNASec in
place of its own tRNASec both in vivo and in vitro
[125]. It may well be that tRNASec is functionally conserved
between the different domains of life despite bacteria using a different tRNA-dependent
route for Sec formation than Sec-decoding archaea and eukaryotes.
Sep-tRNA: Sec-tRNA Synthase
SepSecS Catalyzed Sec-tRNASec Formation
While SepSecS catalyzes a similar reaction as SepCysS, a
tRNA-dependent b-replacement of Sep, a structural phylogeny revealed that SepSecS is
not closely related to SepCysS nor other PLP-dependent enzymes [126]. The
recently completed crystal structures of the SepSecS from M. maripaludis
[126] and mouse [127] to high resolution have enabled insight into how the
enzyme catalyzes the tRNA-dependent formation of Sec. SepSecS forms an (a2)2 homotetramer, mediated by an N-terminal extension in SepSecS. Each
dimer has two active sites, each formed by conserved residues from both subunits
in the dimer interface (Fig. 7) [126,127]. Interestingly deleting the
N-terminal extension, thus apparently disrupting dimerization, gives rise to
inactive SepSecS [126]. Tetramerization is speculated to also enable formation
of large patches of positive electric potential on the surface of the tetramer,
which are predicted to be tRNASec binding sites [127].As in the SepCysS active site, a conserved Asn in the SepSecS (247, M.
maripaludis numbering) active site binds to the nitrogen of the ring
structure of the PLP and similar to other PLP-dependent enzymes a conserved Lys
(278, M. maripaludis numbering) forms a Schiff base linkage with the
co-factor [126]. The phosphate group(s) of Sep-tRNASec and/or
selenophosphate are proposed to interact with conserved Arg, Gln, and Ser in
the active site of SepSecS [126]. Mutations to those residues results in mutant
enzymes with significantly reduced activities both in vitro and in
vivo [126]. SepSecS may exclude free amino acids including Sep from its
active site by using a conserved Glu, which could repel the carboxyl group of
free amino acids [127]. Unlike SepCysS, a conserved Cys residue is not found in
the active site of SepSecS, suggesting that the formation of a perselenide
intermediate is unlikely [126].
Relationship between
tRNA-dependent Cys and Sec biosynthesis
The tRNA-dependent route for Cys biosynthesis is similar to that for
Sec-tRNASec formation in archaea and eukaryotes, since Sep-tRNA serves as the
final precursor prior to product formation in both of them. Both pathways were
present in LUCA [7,66]. Interestingly, SepSecS can use thiophosphate in
vitro to form Cys-tRNASec [126] instead of
selenophosphate to synthesize Sec-tRNASec. Numerous homologs of
selenoproteins are found in nature, which possess Cys in place of Sec. Given
that and the similarity between Sec and Cys codons (UGA and UGY, respectively),
it is interesting to speculate that a dynamic relationship has existed between
the two amino acids over the course of evolution [128].
Outlook
The tRNA-dependent pathways forming Gln-tRNAGln and
Asn-tRNAAsn through amino acid transformations are thought to have evolved
earlier than the direct aminoacylation of the tRNAs with their cognate amino
acids [9,60–63]. In the case of Cys-tRNACys
formation, both the direct and the indirect pathways have been shown to be
present in the time of LUCA [66,75,77]. Regarding Sec, Sec-tRNASec is formed in all domains of life only through the indirect
tRNA-dependent amino acid transformations [100–102]. Even though it cannot
be generalized that indirect pathways are ancient pathways, it does appear that
indirect pathways have unique features that have been retained throughout
evolution. For example, tRNA-dependent Sec-tRNASec formation
may provide a solution to discriminate against Cys, an extremely similar amino
acid, and maintain a faithful translation. A common feature among these indirect pathways is the existence of
misacylated intermediates, which would drastically decrease the fidelity of
translation if they participated in decoding. Even though elongation factors
bind misacylated intermediates with too low or too high affinity in vitro [21,48,50,129–131], it is
still ambiguous whether the discrimination by the elongation factor alone could
ensure the accuracy of translation in vivo [51,132]. Substrate
channeling provides an additional mechanism to prevent misincorporation.
Interestingly, complexes consist of enzymes in the same tRNA-dependent pathway,
and the corresponding tRNA molecules have either been observed or proposed
based on computer modeling [34,51,78]. Furthermore, complex formation may
increase the overall reaction efficiency as well as the stability of the end
product [51,52]. The tRNA-dependent amino acid transformations couple
translation with the biosynthesis of amino acids, which may be involved in
other biological pathways [64]. To better understand the connection and
regulation among different biological processes, systems biology is likely to
be a very useful approach, and it may also lead to discovery of other exciting
aspects of tRNA-dependent amino acid transformation pathways.
Acknowledgements
We would like to thank all the current
members of Dr. Dieter S?ll’s laboratory for discussing the manuscript and assisting
with its revision. We would also like to thank Drs. T. Suzuki, J. Alfonzo and
W.B. Whitman for providing some of their unpublished results.
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