The 3D Structure of Thermus thermophilus Asparaginyl-tRNA Synthetase

Among AsnRSs of various origins assayed for crystallization, only the enzyme from T. thermophilus gave crystals suitable for analysis.¹⁵ This agrees with the remarkable ability of thermostable proteins to crystallize.²⁴ The enzyme expressed in E. coli crystallized in polyethylene glycol 6000 solutions and its structure was solved from isomorphous unanium derivatives of crystals diffracting at 2.6 Å of resolution.^15,16 Soaking of these crystals in ATP•Mg²⁺ solution provoked disordering of the P6₄22 crystal and resulted in a new crystal form with a P6₅22 hexagonal space group. In this crystal, the dimer becomes the asymmetric subunit, as a probable consequence from asymmetric binding of ATP on each subunit. This crystal reverted to the initial form diffracting at 2.95 Å when the ATP sites from both subunits were fully occupied. In contrast to other class II aaRSs, the three Mg²⁺ ions were clearly localized, probably because of the low salt concentration of the crystallization medium. Cocrystallization of the enzyme with ATP, Mg²⁺ and Asn resulted in obtention of crystals containing asparaginyl-adenylate (Asn∼AMP) diffracting only at 3.2 Å.¹⁶ Finally the structure of AsnRS complexed to the activated aa was solved from crystals containing the non hydrolyzable 5'-O-(N-asparaginyl-sulfamoyl)adenosine analog diffracting at 2.65 Å.¹⁶

The modular organization of each subunit resembles that of AspRS and LysRS and consists in a Nt β-barrel connected by a small hinge region to the larger C-terminal (Ct) domain built upon an αβ fold (fig. 1).¹⁶

Figure 1

Comparison of the modular organization of the subclass IIb aminoacyl-tRNA synthetases. A-3D structures of T. thermophilus Asn-, Lys- and yeast AspRS. The upper and the lower modules represent respectively the catalytic and the anticodon-binding domains; (more...)

The N-terminal domain (residues 1 to 97) is formed by a five-stranded β-barrel (strands S1 to S5), in which strands S3 and S4 are separated by a α helix (H0). The 3D structures of yeast AspRS²⁵ and T. thermophilus AspRS²⁶ and LysRS²⁷ complexed with the tRNA indicate, by analogy, that this domain binds the tRNA anticodon. This β-barrel which characterizes subclass IIb aaRSs is functionally equivalent to the mixed αβ fold located in the C-terminus of subclass IIa aaRSs, except for seryl-tRNA synthetase deprived of this domain. The Nt segment, preceding strand S1, and of variable length in subclass IIb aaRSs, is significantly longer in eukaryotes than in prokaryotes. It includes a 3₁₀ helix (G1) of 7 residues in the thermophilic enzyme.

The hinge region (residues 98 to 123), formed by a turn followed by two a helices (H1 and H2), connects the Nt to the Ct domain. This region resembles structurally that of LysRSs whereas in AspRSs, the longer and more globular domain contains additional helices.

The C-terminal domain (residues 124 to 438) is built upon a αβ fold including the three class-defining motifs. It displays the catalytic site formed by a six-stranded antiparallel β-sheet (A2, A3 and A5 to A8) and by the dimer interface which includes motif 1. This β-sheet is interrupted between motifs 2 and 3 by an insertion of 62 residues (256 to 318) in AsnRS from T. thermophilus. This module, inserted between helices H7 and H8, is formed by four helices (H6 to H9) and a β-strand (A4). The topology of this extra-domain resembles that of eukaryotic AspRSs which is significantly smaller than in prokaryotic AspRSs. The interfaces of helices H3, H5 and H6 contain interacting Asp, Glu and Arg residues forming ion pair clusters which contribute to thermal stability of the thermophilic AsnRS. Such type of stabilization has not been reported for T. thermophilus AspRS and LysRS, but for other thermophilic enzymes.²⁸

Despite strong resemblances of their modular organization, superposition of the catalytic domains of class IIb aaRS reveals angular shifts of the Nt domain which can reach up to 18.8°.^16,27,29 (fig. 1).

Contacts between the subunit interfaces. Association of the subunits involves four types of interactions which are mostly preserved in AspRS and LysRS. (i) On the top of the interface, contacts between the Ct domains involve residues from motif 1 distributed along the interfaces of H3 α-helices and the following A1 strands. These interactions, essentially polar, involve Asp, Glu and Arg residues but are reinforced by stacking of the His142 residues from each subunit. (ii) On the bottom of the dimer interface, β-strands I1 and I2 from each monomer form an antiparallel β-sheet. (iii) The internal parts of the interfaces are associated by hydrophobic interactions involving Ile, Leu and Val residues from strands A1, I1 and I2, and Phe 207 of motif 2 from each subunit. (iv) Finally, association is strengthened by inter-subunit contacts between the Ct domain from one subunit and the Nt and hinge domains from the other subunit.

Interaction of Asparaginyl-tRNA Synthetase with ATP•Mg²⁺, Asparaginyl-Adenylate and its Analog

Subunit interactions link intimately the dimer interface, in particular the conserved motif 1, with the ATP-binding site. ATP interacts as in the other class II aaRSs with the expected contribution of the conserved residues from motifs 2 and 3 (Table 1). Interactions of aa residues with the adenine ring and the γ-phosphate stabilize ATP in the bent-conformation, essential for aa activation by class II aaRSs. Three Mg²⁺ ions are associated with the triphosphate group. Mg²⁺1 binds to α and β phosphates and to Asp352 and Glu361, two residues conserved in class II aaRSs (Table 1). Mg²⁺ 2 and 3 interact with phosphates β and γ, one on each side from phosphoester bond.

Table 1

Amino acid groups of AspRS and AsnRS from T. thermophilus contacting the chemical groups of aa∼AMP.

Cocrystallization of AsnRS with ATP•Mg²⁺ and Asn leads to formation of Asn∼AMP bound in the active site in an elongated conformation with Mg²⁺1. The protein residues contacting the substrate groups were identified with the 5'-O-(N-asparaginylsulfamoyl) adenosine analog. The adenosine moiety interacts as in ATP (Table 1 and fig. 2A). The α-amino group of Asn is positioned by Glu164, Ser185 and Gln187, whereas the carbonyle and the amide groups make hydrogen bonds with Arg368 and Glu225, respectively (fig. 2A).

Figure 2

Schematic representation of recognition of aminoacyladenylate by AsnRS and AspRS. A-Interaction of AsnRS with Asn∼AMP. B-Interaction of AspRS with Asp∼AMP. Residues on black, grey and white backgrounds are respectively not conserved, or (more...)

Comparison of the 3D structures of free AsnRS with that complexed with Asn∼AMP shows that binding of the activated aa promotes domain movements in the protein resulting in changes of the enzyme conformation and a more precise delimitation of the catalytic site. Motif 2 loop and the loop preceding β-sheet I1, become more ordered. The first moves in a concerted way with the Ct peptide towards the active site, whereas the latter swings over the catalytic site closing its access and sequestering the activated Asn. The novel interactions shift the two helices H10 and H11 towards the active site and promote rotation by 2.5° of the Nt β-barrel relative to the catalytic domain. This conformation is stabilized by inter-subunit interactions between residues of helix H10 of one subunit and the Nt and hinge domains from the other subunit. This sequence of events, induced by formation of aa∼AMP, was also reported for the other subclass IIb aaRSs.^27,29,30

Exposure of motif 2 loop on the enzyme surface and its contribution in stabilization of the complex with the small ligands are supported by experimental evidences. (i) Limited trypsic digestion of E. coli AsnRS generates a stable peptide of Mr 26,000 starting with residue His243 located in this loop.¹² (ii) Escherichia coli mutant HO202, in which Pro231 from motif 2 (residue 439 from alignment) is replaced by Leu, exhibits a temperature-dependent increase of the K_M for Asn and ATP in ATP-PPi exchange without significant alteration of the k_cat.⁹ The reversible effect of this mutation indicates that Pro231 is not directly involved in substrate binding but more likely in stabilization of the complex and suggests that this residue participates in the positioning of the loop formed by motif 2.

Comparison of Asparaginyl-tRNA Synthetases from Various Phylae and Similarities with Aspartyl-tRNA Synthetases

Figure 3 shows multiple sequence alignments of AsnRSs from all domains of life. Two representatives from each phylum are presented, but the consensus sequence derives from alignment of the 46 known AsnRSs. The modular organization described for the T. thermophilus enzyme is conserved in all AsnRSs. The Nt-, hinge-, and Ct domains, include respectively residues 1 to 272, 273 to 321 and 322 to 921 from consensus sequence. Thirty-four residues are strictly conserved, respectively 4, 1 and 29 in the Nt-, hinge- and catalytic domains. About 122 residues are conserved in 80% of AsnRSs. Most conserved residues are involved in substrate binding or in intra/inter-domain interactions. Thirty of the conserved or semi-conserved residues are also conserved in AspRSs. Table 1 and Figures 2 and 3 summarize the role of the conserved residues in ATP and aa∼AMP binding as revealed by comparison of the 3D structures of AsnRS and AspRS complexed with the ligands.^16,30 The role of these residues in AsnRS has, until now, not been investigated by site-directed mutagenesis. The role of only two of these residues has been defined. In yeast strain YHRO19 auxotroph for Asn, which permitted cloning of the AsnRS gene by complementation, Gly479 from motif 3 of AsnRS is substituted by a Ser residue. It was shown that replacement of this conserved residue, adjacent to residues essential for adenylate binding, increases 10 fold K_M for Asn.³¹ Further, substitution in E. coli AsnRS of Tyr426, a residue at the beginning of motif 3, by Ser increases K_M for ATP, suggesting implication of this residue in ATP binding.³²

With the exception of the appended Nt extension common to all eukaryotic aaRSs (108 to 116 residues in eukaryotic AsnRSs), the alignment does not reveal phylum-specific insertions or extensions in AsnRSs. This contrasts with AspRSs that exhibit species-specific structural signatures.³³ The Nt extension, larger in AsnRSs than in AspRSs, contains 9 to 19% of Lys residues. However, Lys-rich sequences were also found in the appended domain of other eukaryotic aaRSs. Experimental evidences have shown implication of the Nt extension in formation of a high molecular weight complex involving AspRS and 8 other aaRSs in metazoans.³⁴ However, AsnRS is found only in free form, excluding such role for its extension. Moreover, presence of an Nt extension in AspRS and AsnRS from yeast, lacking aaRS complexes, argues further for another role for this domain. Modelization and NMR investigations have shown organization of the Lys-rich sequence of yeast and mammalian AspRSs in an amphiphilic α-helix.^35,36 Recent results suggest involvement of this fold, probably conserved in eukaryotic AsnRSs, in tRNA binding and in stabilization of the complex with the enzyme. It has been shown that in yeast AspRS, the Lys-rich sequence (residues 32xSKxxLKKxxK42 boxed in the consensus sequence in fig. 3) together with other residues from Nt domain, binds tRNA by the minor groove of the anticodon stem, and increases its affinity for the enzyme.³⁷ Since the consensus sequence is partly conserved in LysRSs and in AsnRSs (fig. 3), this domain may be implicated in selection and recognition of tRNA by aaRSs of subclass IIb. Variable position of this sequence in the Nt extension of the three enzymes might be related to different orientation of the anticodon-binding domain with respect to the catalytic domain (fig. 1).¹⁶

Figure 3

Comparative alignments of AspRS and AsnRS. The alignment of 53 AspRS and 46 AsnRS is summarized. Sequences of only two AsnRS from each kingdom are detailed. Percentage of homology is symbolized by shading: black and dark-gray shadings denote 100% and (more...)

Structural Basis for the Discrimination between Asp and Asn by Asparaginyl-tRNA Synthetase

Superposition of AsnRS and AspRS catalytic sites complexed with their homologous aa∼AMP shows that most residues contacting ATP and aa are identical,^16,30 and reveals how discrimination between Asp and Asn can occur (fig.2A,B). Specific recognition is directed by a distinct structural context in the two aaRSs related to 3 systematic differences found in all AspRSs and AsnRSs¹⁶. (i) A Lys residue conserved in AspRS (Lys204 in T. thermophilus AspRS) is replaced by a small or an uncharged residue in AsnRS (Ala 190 in T. thermophilus AsnRS). (ii) This residue is followed by Gln in AspRS and Glu in AsnRS (Gln205 and Glu191 in T. thermophilus AspRS and AsnRS). (iii) An invariant Asp in motif 2 of AspRS is substituted by Glu in AsnRS (Asp239 and Glu225 in T. thermophilus AspRS and AsnRS).

Interaction of AspRS with Asp is based on the electrostatic complementarity between the negatively charged β-carboxylate group of Asp and the positively charged side-chains of Arg and Lys residues (Arg483 and Lys204 in T. thermophilus AspRS), which are positioned by the carboxylate groups of Glu241 and Asp239 (fig. 2B). This structural context disfavours interaction of the carboxyamide group of Asn which would lead to an unfavorable head-on interaction with the Lys and Arg residues. Conversely, AsnRS lacks the Lys which is substituted by a small side-chained residue (Ala 190), but displays the conserved Arg (Arg368) in the same configuration. However, the unfavorable head-on interaction of the carboxyamide of Asn with the Arg residue is avoided by a 60° rotation of the C-Cα bound which turns the Asn side chain more into the catalytic site. As a consequence, the carbonyle and the amido groups of Asn are hydrogen bounded to Arg368 and the adjacent Glu225 (fig. 2A). The latter residue plays the dual role of recognition of Asn and rejection of the negatively charged β-carboxylate group of Asp. The rotation of the side chain of Asn implies a displacement of its α-amino group which is precisely positioned by three hydrogen bonds with conserved Ser185, Gln187 and Glu164 residues (fig. 2A).

In AspRS, Lys204 fulfils the reverse dual role of Glu225 in AsnRS, by interacting with the negatively charged β-carboxylate of Asp and discriminating against Asn. This residue is positioned by Asp239, shorter than its equivalent Glu225 in AsnRS, and is thus too distant from Asp side chain to provoke electrostatic repulsion (fig. 2B). The residues which in AsnRS orient Asn side chain, are conserved in AspRS (Ser199, Gln201, and Glu177), but subtle changes in the position of their side-chains induce a different orientation of the α-amino group of Asp. In conclusion, a limited number of residues differ in the catalytic sites of the two enzymes and are directly implicated in selection of the homologous aa, but other residues participate additionally in correct orientation of the substrates side-chains and are indirectly involved in their selection.

Organellar Asparaginyl-tRNA Synthetases

Mitochondrial and cytosolic AsnRSs from yeast are encoded by distinct nuclear genes, like for 17 other aaRSs in this organism.³⁸ As a consequence, disruption of the gene encoding the mitochondrial enzyme induces a ‘petite’ phenotype but does not affect cell viability. The 14 Nt residues of the mitochondrial yeast enzyme constitute the targeting signal that is removed by proteolytic cleavage upon import.³⁸

The genome of A. thaliana encodes 4 AsnRSs, one of which contains an Nt peptide of 71 residues with overlapping mitochondrial (51 aa) and chloroplastic (63 aa) targeting sequences.^39,40 Green fluorescent protein (GPF) fusion experiments demonstrated import of this AsnRS in chloroplasts and mitochondria. Phylogenetic analyses show that the 4 AsnRSs are clearly distinct from other eukaryotic cytosolic AsnRSs, suggesting that they evolved by repeated duplication of a gene transferred from an ancestral plastid. They constitute an example of capture of an organellar aaRS by the cytosolic protein synthesis machinery.

Involvement of Asparaginyl-tRNA Synthetases in Immune Reactions

AsnRS from the human filarial parasite Brugia malayi, which causes lymphatic filariasis, was shown to be a protective immunodominant antigen.^41,42 It was later shown that sera from patients with interstitial lung disease and inflammatory arthritis contain antibodies which very specifically inhibit AsnRS and precipitate the synthetase complexed with tRNA.^43,44 This was the sixth example of a human aaRS involved in autoimmune reactions. Analysis of the effect of the autoantibodies on AsnRS deletion mutants allowed identification of two distinct epitopes.⁴⁵ A first one located in the Nt domain (residues 1-221) was identified by Western-blot and is not involved in enzyme inactivation. A second one localized in the Ct domain (residues 108-548), probably in the catalytic β-barrel and/or in the hinge region, is involved in enzyme inactivation. The antibodies are without effect on aa activation but inactivate tRNA charging and, surprisingly, increase affinity of the enzyme for tRNA. Thus, enzyme inactivation might be provoked by blocking either dissociation of the charged tRNA, or the conformational changes promoting formation of the competent enzyme•tRNA complex. This effect contrasts with that described for other aminoacylation systems where inactivation by autoantibodies was related to inhibition of aa activation or to precipitation of the cognate tRNA. The physiological processes involved in production of autoimmune phenomena are not understood. It has been proposed that autoantibodies arose through an immune response to foreign antigens such as infectious agents that share, by molecular mimicry, common structures with host proteins.⁴⁵

Structural and Functional Peculiarities of tRNA^Asn

The various organisms contain one tRNA^Asn with anticodon GUU able to recognize the AAU and AAC codons encoding Asn. Sixteen tRNA^Asn of various origins are sequenced.⁴⁶ tRNA^Asn belongs to class 1 tRNAs characterized by a small variable-loop containing 5 or exceptionnally, in organellar tRNA^Asn, 4 nucleotides. Prokaryotic and eukaryotic tRNA^Asn contain mostly respectively 76 and 77 nucleotides, because of variations in their D-loops containing 8 or 9 nucleotides. However, the D-loop of Methanobacterium thermoautotrophicum tRNA^Asn contains 10 nucleotides and that of mitochondrial tRNA^Asn displays 3 (e.g., in bronchiostoma) to 11 (in albinoni coerulea). The D-loops of eubacterial tRNA^Asn contain two dihydrouridines (D) whereas archaebacterial tRNA^Asn are deprived of this modification. In contrast, D-loops of eukaryotic tRNA^Asn contain four modified U: four D are present in yeast tRNA^Asn and three D in addition to 3-(3-amino-3-carboxypropyl)-Uridine (acp³U) in mammalian tRNA^Asn. The latter modified U is also present in the D-loop of M. thermoautotrophicum tRNA^Asn. D is also found on position 47 of the variable-loop of eukaryotic tRNA^Asn and pseudouridine (F) in positions 27 and 28 of the anticodon-stem in mammalian tRNA^Asn. In contrast to other tRNA species, no atypical secondary structures were found in mitochondrial tRNA^Asn.

Two unusual modifications are found in tRNA^Asn: queuine at position 34, the first nucleotide from anticodon, in eukaryotic and most eubacterial tRNA^Asn and N-((9-β-Dribofuraanosylpurine- 6-yl)carbamoyl)threonine (t⁶A) at position 37 in almost all tRNA^Asn. These modifications can, however, also be present in other tRNA species. Queuine, a modified guanine, is inserted by exchange with G₃₄ by a tRNA-guanine transglycosylase.⁴⁷ Certain eubacterial subgroups such as Thermus and all archaea are unable to synthesize queuine. In these species, tRNA^Asn contains G₃₄. tRNA^Asn from T. thermophilus like the other tRNAs sequenced from this organism contain 3 invariant post-transcriptional modifications: Gm₁₈, s²T₅₄ and m¹A₅₈ (see ref. 33 and refs. therein). It has been shown that these modifications are not involved in the functionality of the tRNAs, but they reinforce their thermal stability. ^10,48 Finally T. themophilus tRNA^Asn contains m²G at positions 6 and 10. These modifications were until now not found in other prokaryotic tRNA^Asn. In contrast m²G₁₀ and m²₂G₂₆ are present in eukaryotic tRNA^Asn and additionally m¹G₉ in mammalian tRNA^Asn.

Cross-reactions with tRNA^Asn and AsnRSs of various origins reveal species specificity in tRNA asparaginylation. Human and B. malayi AsnRSs charge well mammalian and yeast tRNA^Asn but poorly E. coli tRNA^Asn,^31,42,44 whereas AsnRS from T. thermophilus aspartylates efficiently T. thermophilus and E. coli tRNA^Asn but only poorly yeast tRNA^Asn. ¹⁰ It has been suggested that species specificity of AsnRSs is related to the additional base inserted in position 21 of the D-loop in eukaryotic tRNA^Asn.⁴⁴

Identity of tRNA^Asn

The nucleotides defining Asn identity and the possible implication of post-transcriptional modifications in tRNA recognition by AsnRS have been investigated in prokaryotic systems.^49,50

Eubacterial tRNA^Asn all begin with 5'pU, preventing efficient transcription with T7 RNA polymerase. However, the tRNA^Asn transcript from Thermus thermophilus could be obtained by self-cleavage of a large transcript in which tRNA^Asn was extended at the 5' end by a hammerhead ribozyme. This unmodified tRNA^Asn is charged only 6-fold less efficiently by AsnRS than the modified tRNA^Asn isolated from T. thermophilus.¹⁰ Thus, the post-transcriptional modifications, are not crucial for asparaginylation, although they exert a limited effect on its charging efficiency.

Because of the difficulty to obtain tRNA^Asn transcripts, Asn identity has not been investigated by analysis of the charging capacity of variants created by in vitro transcription of the mutated gene. Also, in vivo approaches, using variants of either initiator tRNA transplantated with the Asn anticodon or tRNA^Asn suppressor failed. Indeed, transplantation of the Asn anticodon into tRNA^fMet did not lead to initiation of protein synthesis from an Asn codon.⁴⁹ Further, conversion of tRNA^Asn into amber or opal suppressors, through change of the anticodon, inactivates the tRNA, and it was not possible to restaure its aminoacylation capacity by compensatory mutations in the anticodon stem and loop like for tRNA^Asp.⁵¹ In fact, random mutagenesis in yeast tRNA^Asn(CUA) creates variants charged by GlnRS.⁵² Acquisition of Gln specificity by the tRNA^Asn amber suppressor containing already several Gln identity elements is promoted by creation of mutations triggering disruption of the first base-pair from acceptor stem, essential for tRNA glutaminylation. Nevertheless, these studies showed that the anticodon of tRNA^Asn is an essential element in Asn identity. Additionnal informations about implication of the anticodon nucleotides and other elements from the tRNA in Asn identity were obtained by analysis of variants of E. coli tRNA^Lys(UUU) transcribed in vitro and E. coli tRNA^Asn(GUU) expressed in vivo.^49,50

The primordial role of the first base from anticodon (G₃₄) was revealed by switch of the specificity of a tRNA^Lys transcript transplantated with both the anticodon and the discriminator base of tRNA^Asn (GUU and G₇₃ instead of UUU and A₇₃). This chimeric tRNA^Lys(GUU) becomes well charged with Asn.⁵⁰ Since tRNA^Lys(UUU) with a G₇₃ does not accept Asn, G₃₄ plays a crucial role in Asn specificity. However, the discriminatory G is also important, since the presence of any other nucleotide on this position decreases asparaginylation efficiency of this tRNA.

The contribution of the anticodon nucleotides to Asn identity was more extensively investigated by in vivo analysis of the charging capacity of tRNA^Asn variants.⁴⁹ Charged and uncharged tRNA were fractionated by PAGE and quantified by Northern blot. Single base changes of U₃₅ to C₃₅ or U₃₆ to C₃₆ in tRNA^Asn abolish its charging capacity whereas substitution of G₃₄ by C₃₄ converting the anticodon GUU to CUU confers Lys accepting capacity to the mutated tRNA. Finally, the chimeric tRNA^Asn (CUU) with A₇₃ is deprived of any Asn accepting capacity and switches, in vivo, to Lys specificity. Thus, the anticodon nucleotides G₃₄, U₃₅ and U₃₆ as well as the discriminator G₇₃ are crucial for asparaginylation (fig.4). In addition to its functional role, G₇₃ plays also a pivotal role by maintaining the overall structure of tRNA^Asn since presence of C₇₃ or U₇₃ provokes in vivo instability. These nucleotides are probably the major but not the sole elements determining Asn identity, since the chimeric tRNA^fMet with transplantated Asn anticodon and G₇₃ does not initiate synthesis of a polypeptide chain from an Asn initiation codon.⁴⁹

Figure 4

tRNA^Asn identity and putative interaction of U₃₅ and G₇₃ with AsnRS residues. The cloverleaf structure of E. coli tRNA^Asn is presented. Nucleotides determining E. coli Asn identity are indicated in grey dots. Parts of the consensus sequences deduced from (more...)

Discrimination by Asparaginyl-tRNA Synthetase of tRNA^Asn against tRNA^Asp and tRNA^Lys

The strongest identity elements of tRNA^Asn, namely the anticodon nucleotides and the discriminator base are partly conserved in tRNA^Asp53-56 and tRNA^Lys.^57-60 Then, how does AsnRS discriminate tRNA^Asn from tRNA^Asp and tRNA^Lys? Among the nucleotides involved in identity of tRNA^Asn and tRNA^Asp only nucleotides 36, (respectively C and U) differs. Since substitution in E. coli tRNA^Asn of U₃₆ by C₃₆ drastically decreases charging efficiency,⁵⁰ C₃₆ prevents recognition of tRNA^Asp by AsnRS. In contrast, U₃₆ in tRNA^Asn by itself probably does not prevent aspartylation by AspRS since substitution in tRNA^Asp of C₃₆ by U₃₆ only affects moderately tRNA aspartylation.⁵⁶ Specific asparaginylation of tRNA^Asn may be reinforced by the postranscriptionnal modification t⁶A preventing aspartylation.

Among the nucleotides involved in identity of tRNA^Asn and tRNA^Lys(UUU) only nucleotide 34, (respectively G and hypermodified U) differs in cytosolic eukaryotic tRNAs, whereas both, nucleotide 34 and the discriminator base (respectively G₇₃ and A₇₃), differ in prokaryotic tRNAs. So, in eukaryotic tRNA^Lys(UUU) the modified U₃₄ is probably the sole nucleotide which prevents asparaginylation, whereas in prokaryotic tRNA^Lys(UUU) the discriminator base A₇₃ may also prevent asparaginylation.

Finally it has been suggested that strong contribution of U35 and U36 to Asn identity determines the distribution of the two distantly related groups of class I LysRS which recognize either both U35 and U36 or U36 alone, in organisms possessing or lacking AsnRS.⁶¹

Elements of Asparaginyl-tRNA Synthetase Involved in Recognition of tRNA^Asn

The aa residues of AspRS contacting the anticodon and the discriminator base of tRNA^Asp were identified in the 3D structure of the complex from yeast.^25,62 Implication of most of these residues in efficient tRNA charging was confirmed by site-directed mutagenesis of the enzyme.⁶³ Alignments show conservation in AsnRS of the residues from AspRS contacting U₃₅ and G₇₃ of tRNA^Asp.^54,64 Since these nucleotides are conserved in tRNA^Asn and contribute also to efficient asparaginylation they are probably contacted by the conserved residues from AsnRS (fig. 4).

Characterization, General Properties and Occurrence

Biochemical and genomic data reveal the existence in various prokaryotes of a non-conventional pathway of tRNA asparaginylation. This pathway was first discovered in Haloferax volcani.^21,65 It was shown that in this archaea, deprived of AsnRS, Asn-tRNA is formed by amidation of Asp bound on tRNA. This suggested indirect synthesis of Asn-tRNA^Asn by a pathway resembling that previously described for formation of Gln-tRNA^Gln.²³ Further informations about this pathway, were obtained by analysis of Asn-tRNA^Asn synthesis in T. thermophilus. This thermophilic eubacterium possesses two AspRS (AspRS1 and AspRS2).⁶⁴ Alignments show that AspRS1 resembles structurally eubacterial AspRS, and AspRS2, of significantly shorter size, resembles archaeal AspRS.³³ AspRS1 is discriminating and aspartylates only tRNA^Asp, whereas AspRS2, non-discriminating, aspartylates tRNA^Asn in addition to tRNA^Asp, with similar efficiencies (reaction 1).²² Asp-tRNA^Asn formed by AspRS2 is then converted into Asn-tRNA^Asn by an tRNA-dependent aspartyl-tRNA^Asn amidotransferase (Asp-AdT) in the presence of an amide group donor (Asn, Gln or ammonia) and ATP (reaction 2, fig. 5).

Figure 5

Interrelation between tRNA aspartylation and asparaginylation in T. thermophilus. The direct and indirect routes of Asn-tRNA formation are shown respectively on light and dark grey.

Reaction 1:

This pathway resembles the indirect route of Gln-tRNA^Gln formation discovered in B. megaterium²³ and later described in other Gram + eubacteria, the Gram-eubacterium Rhizobium meliloti, cyanobacteria, archaea, and most organelles.^66-68 In these organisms deprived of GlnRS, Gln-tRNA^Gln is formed by amidation of Glu mischarged on tRNA^Gln by a GluRS able to attach Glu on tRNA^Gln as efficiently than on tRNA^Glu. Biochemical and genomic investigations reveal that absence of GlnRS is widespread among eubacteria and ubiquitous in archaea, whereas absence of AsnRS is widespread in archaea but exceptionnal in eubacteria⁶⁹ (Table 2). Therefore, prokaryotes use the indirect pathway much more frequently for glutaminylation than for asparaginylation of tRNA⁶⁹. In contrast, eukaryotes form Gln-tRNA^Gln and Asn-tRNA^Asn directly by GlnRS and AsnRS. Organelles possess AsnRS and use the direct pathway for tRNA asparaginylation. However, they are deprived of GlnRS and glutaminylate tRNA by the indirect pathway; the sole known exception is presence of GlnRS in mitochondria from Leishmania tarentolae.⁷⁰

Table 2

Prokaryotes using the direct or/and the indirect route for formation of Asn-tRNA.

The indirect pathway involves formation of Asp-tRNA^Asn whose participation in protein synthesis would be lethal. It was shown that Asp-tRNA^Asn is not vehiculated to the ribosome because it cannot bind to the elongation factor Tu (fig. 5).²² Absence of binding of mischarged Glu-tRNA^Gln, involved in organellar tRNA glutaminylation, to elongation factor Tu from chloroplasts from Pisum sativum has also been reported.⁷¹ Therefore, the partners of the translation machinery adapted their function to the formation of the mischarged tRNA to improve accuracy. The structural basis for the rejection, by the elongation factor Tu, of mischarged aa-tRNA carrying a physiological function remains still unknown.

Coexistence of Direct and Indirect Pathways of tRNA Asparaginylation

Most species using the indirect route for tRNA asparaginylation and all glutaminylating indirectly tRNA are deprived of respectively AsnRS or GlnRS and thus are unable to form directly the homologous aa-tRNA. However, T. thermophilus and Deinococcus radiodurans can asparaginylate tRNA directly and indirectly, since AsnRS coexists with the partners of the indirect pathway.^22,72 Duplication of the pathways of Asn-tRNA synthesis could be rationalyzed by biochemical and genetic analyses showing absence of asparagine synthetase (AS) in these organisms. Under Asn starvation, tRNA asparaginylation occurs by formation of Asn on tRNA^Asn whereas, when the organism is supplied with free Asn, Asn-tRNA^Asn is formed directly by AsnRS. Further, in vitro analysis of the efficiencies of the two pathways shows faster asparaginylation of tRNA directly by AsnRS than indirectly.²² Thus, the direct pathway is conserved, despite absence of its autonomy, since it provides the organism more efficiently with asparaginylated tRNA than the indirect pathway. However, the latter pathway is essential for survival of the organism since it constitutes the unique route of Asn-tRNA^Asn formation in the absence of free Asn (fig. 5).

The Partners of the Indirect Pathway of tRNA Asparaginylation

tRNAAsp and tRNAAsn Recognitions by Aspartyl-tRNA Synthetase of Relaxed Specificity

Since AspRS1 and AsnRS discriminate tRNA^Asp from tRNA^Asn and AspRS2 does not, one may predict that AspRS1 and AsnRS recognize peculiar elements in the cognate tRNA, whereas AspRS2 recognizes elements common to both tRNAs. The elements of tRNA^Asp from T. thermophilus involved in recognition by the two thermophilic AspRSs are presented in Figure 6.⁵⁶ With only one exception, the same nucleotides determine aspartylation by AspRS1 and AspRS2. However, they differ by their contribution. The 3 major determinants for charging by AspRS1 are U₃₅ and C₃₆ from anticodon followed by the discriminator base G₇₃. This triad is followed by the G₂-C₇₁ pair from acceptor stem, the first base from anticodon G₃₄ and finally by C₃₈. The hierarchy of contribution of these elements differs for charging by AspRS2. Contribution of U₃₅ preceds immediately that of the discriminator base G₇₃ which is followed by G₃₄, C₃₈ and finally C₃₆. Therefore, specific charging and selection of the cognate tRNA by AspRS1 and AsnRS is very likely promoted by nucleotide 36 of the anticodon. This base distinguishes tRNA^Asp from tRNA^Asn (respectively C and U) and is essential for charging by the homologous aaRS. Relaxed specificity of AspRS2 is due to the minimized contribution of nucleotide 36 in tRNA recognition.

Figure 6

Relative contribution of tRNA elements to the two aspartate identities of T. thermophilus. Nucleotides determining Asp identity for AspRS1 and AspRS2 are indicated in grey dots and are listed in decreasing order of their contribution (big to small letters). (more...)

The tRNA-Dependent Amidotransferase

The Asp-tRNA^Asn amidotransferase (Asp-AdT) converting Asp mischarged on tRNA^Asn into Asn was first isolated from T. thermophilus in an αβ heterodimeric form.²² Analysis of the genome from T. thermophilus revealed ORF encoding orthologs of GatA, GatB and GatC subunits of the heterotrimeric Glu-tRNA^Gln amidotransferase (Glu-AdT) from B. subtilis.^74,75 Comparison of the Nt sequences showed identity of the α and β subunits of Asp-AdT with GatA and GatB of Glu-AdT, suggesting that both constitute the same enzyme, probably of trimeric aβ? (GatABC) structure.

When the genes encoding GatA, GatB and GatC subunits of T. thermophilus Asp-AdT were cloned in an operon for expression in E. coli, a fully active trimeric enzyme converting Asp-tRNA^Asn into Asn-tRNA^Asn was expressed with a subunit stoichiometry of 1/1/1. This enzyme also converts, in vitro, Glu mischarged on B. subtilis tRNA^Gln into Gln.⁷⁵ Dual specificity was further observed for Glu-AdT of B. subtilis capable to convert, in vitro, Asp mischarged on tRNA^Asn from T. thermophilus or D. radiodurans into Asn.⁷⁵ Finally, orthologs of this enzyme identified in D. radiodurans, Acidothiobacillus ferrooxidans, Chlamydia trachomatis and in the archaeon M. thermoautotrophicum also display dual specificity in vitro.^70,76-77 Thus the trimeric amidotransferase called Asp/Glu-AdT or AdT can ensure in vitro formation of Asn-tRNA^Asn and Gln-tRNA^Gln. However, the cellular function of this enzyme is dictated by the biochemical and genetic background of the host. In the absence of AsnRS (e.g., in H. volcanii) the AdT supplies the organism with Asn-tRNA^Asn, in the absence of GlnRS (e.g., in B. subtilis) with Gln-tRNA^Gln and in the absence of GlnRS and AsnRS (e.g., in A. ferrooxidans and C. trachomatis), with both aa-tRNA.

Despite identical functional properties, important divergences are observed in organization of the AdT genes in the various species. In B. subtilis, A. ferrooxidans and C. trachomatis the genes are organized in an operon (gatCAB), contrasting with their dispersion in the genomes from T. thermophilus, D. radiodurans and archaea.^74-78

Alignment of AdT polypeptide chains shows two interesting features in GatA. (i) The signature sequence of amidases rich in Gly, Ser and Ala (residues 179-210 from consensus sequence), involved in the metabolic pathways of various amino acids (Arg, Pro, Phe, Trp) in procaryotes (e.g., Rhodococcus, Brevibacterium and Pseudomonas species) (fig. 7).^74,77 Inhibition of AdT by boronic acid suggests implication of a Ser residue in catalysis.^79,80 (ii) A P-loop (residues 142-148) which is a structural domain involved in ATP and GTP binding. These features indicate that GatA binds ATP and catalyzes transamidation, and agree with the ability of B. subtilis GatA, when expressed together with GatC, to promote ATP-independent deamidation of Gln.⁷⁴ GatB, structurally related to Pet112, an essential mitochondrial protein, is probably involved in tRNA recognition. Finally GatC might be involved in folding and/or stability of GatA, since in vivo expression of active GatA requires coexpression of GatC.⁷⁴

Figure 7

Alignment of Asp/Glu-AdT sequences from various origins. Only the sequences of T. thermophilus and B. subtilis AdT subunits GatA, B and C are presented. The consensus sequence derives from alignment by clustal X of 40 AdT sequences. Percentage of homology, (more...)

Functional and Structural Interrelations between tRNA-Dependent and tRNA-Independent Amidotransferases and between Amidotransferases and Aminoacyl-tRNA Synthetases

The transamidation catalyzed by AdT resembles mechanistically amidation of free Glu into Gln by glutamine synthetase (GS) since both form a γ carboxyl-Pi intermediate. In contrast, conversion of free Asp into Asn by AS involves formation of a β carboxyl-AMP intermediate. Interestingly aaRS activate their homologous aa by formation of a a carboxyl-AMP. Comparison of the 3D structures of E. coli AS A and B with yeast AspRS reveals a strong resemblance of the catalytic center, in particular conservation of essential aa residues and presence in AS of the ATP-binding motif of class II aaRS.^84,85 The most stricking difference between both structures are related to activation of distinct carboxylate groups of Asn. This observation indicates that AspRS, AsnRS and AS are closely related in evolution. In contrast, binding of ATP by AdT and AS respectively by a P-loop and a motif conserved in class II aaRSs, and presence of the signature motif of amidases in AdTs but not in ASs (fig. 7), argue for a distinct origin of both enzymes and suggest that AdTs and aaRSs are not phylogenetically interconnected. Finally, GS which binds ATP by a non canonical motif and differs structurally from AS and aaRS is evolutionary distant from these enzymes.⁸⁶

Interestingly, AsnRS and AS are related by regulation processes. In L. bulgaricus synthesis of both enzymes is coregulated since their genes are organized in an operon depending on a unique leader region located upstream of AS gene⁸⁷. Evidences have been brougth that transcription of both genes is regulated by a common antitermination mechanism and would be triggered by increase of the uncharged tRNA^Asn in the cell caused by insufficient endogenous Asn.

Glu/Asp-AdT does not constitute the unique tRNA-dependent amidotransferase. In archaea, deprived of GlnRS, tRNA glutaminylation is achieved by a tRNA-dependent amidotransferase converting Glu mischarged on tRNA^Gln into Gln. This heterodimeric enzyme, called Glu-AdT, exhibits, in contrast to Glu/Asp-AdT, a strict specificity and is absent in eubacteria.⁷⁷ However, both enzymes are present in archaea deprived of GlnRS and AsnRS and form respectively Asn-tRNA^Asn and Gln-tRNA^Gln. Presence of a specific Glu-AdT, in addition to Asp/Glu-AdT of dual specificity, which leads to the redundancy of Glu-AdT activity, remains unexplained.

Two Amide Aminoacyl-tRNAs and Two Different Evolutionary Histories

Origin of GlnRS has been confidently established by several phylogenic studies which show that this enzyme arose in eukarya from a duplicate of the gene of GluRS that acquired this new specificity and was transferred to eubacteria by horizontal gene transfer.^88,89 The lack of GlnRS in the archaeal domain and in complete subgroups of eubacteria supports the idea that GlnRS specificity was one of the latest to emerge. This hypothesis is also supported by the theory of coevolution of the genetic code and biosynthesis of the aa,⁹⁰ which postulates that, initially Gln codons encoded Glu as the result of tRNA^Gln charging with Glu by the ancestral GluRS from which originated GlnRS latter in evolution.

Similarly, in primitive systems, Asn codons were likely encoded by Asp as the result of aspartylation of tRNA^Asn by the primordial AspRS. The remarkable extent of structural homologies between AspRSs and AsnRSs lead to the suggestion that AsnRS arose from AspRS by gene duplication and subsequent acquisition of AsnRS specificity by one of the copy. So far, three attempts were made to decipher evolution of the Asp/Asn-tRNA synthetase family and to pinpoint the origin of AsnRS.^91-93 They vary by the pool of AsnRS and AspRS sequences processed and by the phylogenic approaches applied. Each strategy identifies a different kingdom for the origin of AsnRS. Three evolutionary models are currently in competition, which were already proposed by the first and most extensive study (fig. 8).⁹¹ Although conflicting about the origin of AsnRS, these studies agree with emergence of AsnRS prior to GlnRS and evolution of both along different paths. Closer examination of the methods and strategies used to build these phylogenic trees shows that either a limited number of sequences and/or only the catalytic domain of these enzymes were subjected to analysis. We therefore reinvestigated the filiation of AsnRS by using an extended pool of AspRS and AsnRS sequences encompassing both the catalytic and anticodon-binding domains. The tree (fig. 9) was constructed by the Neighbor Joining method from a bootstrap analysis (1000 replicates). This tree shows that AsnRS like AspRS are organized into three clades following the organismal phylogeny. However, bacterial AsnRSs are splitted into two groups: a large one encompassing most of the bacterial AsnRSs and the mitochondrial species (Bact. N1), and a small one constituted of 6 species of firmicutes and T. thermophilus (Bact. N2) and arising from the archaeal AsnRS branch. Although AsnRSs seem to be closer to the eukaryal/archaeal branch of AspRSs in terms of evolution distances, there is no clear indication that these enzymes arose from this subgroup of AspRSs, lesser that eukaryal or archaeal AspRSs have originated them.

Figure 8

The three possible evolutionary trees of Asp/AsnRS family. Asp- and AsnRS are abbreviated by DRS and NRS. Suffixes Bact., Arch. and Euk. refer to the bacterial, archaeal or eukaryotic origins of these aaRSs.

Figure 9

Phylogenic tree of Asp/AsnRS sequences. The tree was deduced by Neighbor Joining analysis from a bootstrap of 1000 replicates based on 53 and 46 AspRS and AsnRS sequences aligned by clustal X.

AsnRS are present in all domains and their phylogenic distribution, with the exception of Bact. N2 species, follows that of AspRS. Topology of AsnRS trees suggests that either AsnRS appeared before separation of the phylae or that early after split of the phylae, the bacterial and the archaeal/eukaryal ancestors independently evolved AsnRS leading to a wide spread of this enzyme among the various organisms, especially among bacteria. Another hypothesis explaining the lack of likelihood between Bact. N1 AsnRS and their eukaryal/archaeal counterparts would be that AsnRS emerged in the latter domain after its separation from bacteria and was immediately transferred into bacteria (from the Bact. N1 type) early thereafter, and evolved independently since then. The small group of Bact. N2 AsnRS did not, however, follow this evolutionary scheme. Their position, arising from the archaeal branch, suggests that their presence in the 7 bacterial species results from a more recent horizontal gene transfer from archaea. Although, existence of an archaeal-type AspRS has already been described, its acquisition has also been correlated to a functional advantage (formation of Asp-tRNA^Asn, substrate of the AdT) for the bacterial host. So far there is no apparent advantage, nor explanation, for the existence of an archaeal-like AsnRS in some bacterial species.

From Aspartate to Asparagine, the Emergence and Evolution Path of a New tRNA Charging Specificity

Based on biochemical and structural data available on Asn-tRNA formation and the consensual phylogenetic arguments produced by studies on AsnRS, we can try to retrace the steps leading to emergence of AsnRS. This evolutionary scenario starts with the assumption that an aaRS displaying the highest degree of specificity for both the aa and its corresponding tRNA is the ultimate stage in the evolution of aa-tRNA synthesis. Conversely, aaRS with relaxed specificities, and formation of aa-tRNA requiring mutiple steps, are hallmarks of lesser evolved enzymes and pathways.

It is admitted that Asn was one of the latest aa introduced in the cell's repertoire for protein synthesis.⁹⁰ In the latest common ancestor, before apparition of Asn, codons specifying Asn (AAU and AAC) were encoded by Asp as the result of the capacity of the ancestral AspRS to acylate tRNA^Asp and tRNA^Asn with Asp. The first step leading to actual incorporation of Asn into proteins, at codons specifying Asn, was apparition of AdT able to convert Asp mischarged on tRNA^Asn into Asn. At some point in the evolution, AsnRS appeared by duplication of the AspRS gene and acquisition by one copy, of the capability to attach Asn on tRNA^Asn. The only restriction to this event relies in the ability of the cell to produce free Asn since existence of an autonomous direct pathway of Asn-tRNA synthesis requires the coexistence of AsnRS and AS. Therefore, acquisition and stabilization of an AsnRS was very likely related to apparition of AS A or B. In most cases, acquisition of the autonomous direct route of Asn-tRNA formation led to loss of the indirect route. However, if AsnRS or AS is missing, organismal synthesis of Asn-tRNA is performed by Asp-AdT (Table 2). In most cases Asp-AdT supplies the absence of AsnRS, or of both AsnRS and AS, but exceptionally only the absence of AS. In these organisms, AsnRS which should be dispensable, has been conserved because it forms Asn-tRNA more efficiently than AdT. However, this selective advantage is only expressed under certain environmental conditions namely when Asn is present.