Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase (original) (raw)

Abstract

The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile ( Mm LeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS ( Ec LeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of Ec LeuRS, can cooperate when inserted into the scaffold of the minimal Mm LeuRS, and this generates an enzyme nearly as active as Ec LeuRS. Further, we show that LSD participates in tRNA Leu recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

INTRODUCTION

Aminoacyl-tRNA synthetases (aaRS) are a large and diverse family of enzymes that catalyze the attachment of amino acids to their cognate tRNAs. Each aaRS specifically attaches its cognate amino acid to its corresponding tRNA isoacceptor. A two-step process is performed by the aaRS during aminoacylation: (i) activation of the amino acid by ATP hydrolysis to form an aminoacyl-adenylate intermediate; (ii) transfer of the aminoacyl moiety from the intermediate to the cognate tRNA isoacceptor to make the aminoacyl-tRNA ( 1 ). Based on sequence homology and the structures of the catalytic active sites, aaRSs are divided into two classes ( 2 ). Leucyl-tRNA synthetase (LeuRS) is a class I aaRS that has an active site folded to form a typical Rossmann dinucleotide-binding fold. According to evolutionary models, the primitive catalytic core of LeuRS was extended by the insertion and appendage of additional domains (also called modules) ( 3 ). Most LeuRSs carry a large insert called the connective polypeptide 1 (CP1) domain that is responsible for the amino acid-editing function. LeuRSs also exhibit tRNA-binding domains that recognize and bind tRNA Leu isoacceptors ( 4–7 ). A well-ordered module inserted into the catalytic domain, named the leucine-specific domain (LSD), is also found in most bacterial and some eukaryotic LeuRSs. LSD is connected to the KMSKS motif via a β-ribbon. The three-dimensional structure of the Thermus thermophilus LeuRS ( Tt LeuRS) shows that the LSD contains five β-strands and two short α-helices ( 3 , 5 ). In comparison, the LSD of Escherichia coli LeuRS ( Ec LeuRS) exhibits an additional extended β-hairpin ( 4 ). Crystal studies have also revealed that the LSD plays a critical role in positioning the conserved catalytic KMSKS loop during aminoacylation reactions ( 4 ).

Although the LSD is mainly found in prokaryotic LeuRSs, it is not highly conserved in sequence or length ( 3 , 4 , 8 ). The heterodimeric αβ-LeuRS from Aquifex aeolicus ( Aa LeuRS) has one of the largest LSDs, and this also serves to split the enzyme into two subunits ( 3 , 9 ) . The LSD can also be missing completely in some species, such as Bacillus subtilis or Mycoplasma mobile , in which LeuRS is remarkable for the complete absence of a CP1-editing domain ( 8 , 10 ) . In addition, sequence alignment has shown that the LSD in the LeuRS from M. mobile ( Mm LeuRS) is replaced by the tetrapeptide 398 GKDG 401 ( 4 , 10 ).

A recent study revealed that the CP1 domain and LSD of Ec LeuRS both undergo large rotations when tRNA shifts from the synthetic site to the editing active site ( Figure 1 A) ( 4 ). The CP1 domain rotates by 12° to open up a passage for the translocation of the 3′ end of the tRNA, while the more dynamic LSD, together with the adjacent catalytically crucial KMSKS loop, is rotated by about 33° between the aminoacylation and editing conformations. Consistently, both the CP1 domain and LSD positions move by about 19° and 35° in the Tt LeuRS when comparing the aminoacylation and editing conformations ( 5 ). Another study indicated that the tRNA-triggered conformational rearrangement leads to inter-domain communication between the editing and synthetic domains of Ec LeuRS ( 11 ). All these data strongly suggest that both the CP1 domain and LSD are functionally connected and cooperate during the aminoacylation and editing reactions.

Figure 1.

Impact of LSD mutations on aminoacylation and editing of Ec LeuRS. ( A ) Three-dimensional view of Ec LeuRS showing the LSD motion in the aminoacylation (blue) and editing state (red) (PDB entry 4AQ7 and 4ARC). ( B ) Sequence alignment based on structural elements of the LeuRS LSD; the tetrapeptide linker is highlighted in green. Ec, Escherichia coli ; Aa, Aquifex aeolicus; Mm, Mycoplasma mobile . ( C ) Aminoacylation of 10 µM Ec tRNA Leu by 5 nM of Ec LeuRS (black circle), Ec LeuRS-GKDG (black square) and Ec LeuRS-AAAA (black triangle). ( D ) Hydrolysis of 1 µM [ 3 H]-Ile- Ec tRNA Leu by 5 nM of Ec LeuRS (black circle), Ec LeuRS-GKDG (black square), Ec LeuRS-AAAA (black triangle) and no enzyme (open circle) .

tRNA Leu , together with tRNA Ser and tRNA Tyr , are class II tRNAs which are characterized by the presence of both a long variable stem and loop ( 12 ). Interactions between LeuRS and tRNA Leu have been extensively investigated, and the conserved A73 nucleotide is considered to be the main element for identification. The amino acid-accepting end (CCA 76 ) of Ec tRNA Leu is critical for both the aminoacylation and the editing processes ( 13 ). The tertiary interactions between the D- and T-loops that determine the tRNA folding are additional critical elements of the leucine identity ( 14 ). In addition, tRNA elements that are critical during the editing process have been detected in the anticodon arms of tRNA Leu from A. aeolicus and Saccharomyces cerevisiae ( 15 , 16 ).

In this present study, the LSD of Ec LeuRS was substituted with the minimal tetrapeptide linker GKDG from the Mm LeuRS, and this created a chimeric mutant named Ec LeuRS-GKDG. In addition, the LSD and CP1 domain of Ec LeuRS were inserted into the minimal Mm LeuRS, and this produced another chimera termed Mm LeuRS-CP1/LSD. By comparing the catalytic performances of these chimeric enzymes, we found that the LSD is essential for neither aminoacylation nor editing functions of LeuRSs. However, LSD participates in tRNA binding, and it is able to discriminate between different tRNA Leu isoacceptors. Indeed, the LSD acts as a sensor that can measure the size of the V-arm loop and identify the nucleotide at position 20 of the tRNA Leu . These results highlight the role of the LSD during tRNA recognition and suggest that interactions between the LSD and tRNA Leu might favour binding in both aminoacylation and editing catalytic steps. Altogether, these results emphasize the modular nature of the LSD as well as the important contribution played by the other synthetase modules in enhancing catalytic efficiency and tRNA specificity.

MATERIALS AND METHODS

Materials

l -leucine, l -norvaline (Nva), ATP, Tris-HCl buffer, MgCl 2 solution and dithiothreitol (DTT) were purchased from Sigma (USA). [ 3 H] l -leucine, [ 3 H] l -isoleucine and adenosine 5′-[α- 32 P] triphosphate were obtained from PerkinElmer Life Sciences (USA). PEI Cellulose F plates for thin layer chromatography (TLC) were purchased from Merck (Germany). T4 DNA ligase and other restriction endonucleases were from MBI Fermentas (Lithuania). DEAE-Sepharose CL-6B and Superdex TM 75 were purchased from GE Healthcare (USA). Ni 2+ -NTA Superflow was purchased from Qiagen, Inc. (Germany). Plasmid pET30a was obtained from Novagen (USA), and E. coli strain BL21 (DE3) was from Invitrogen (USA). The expression vector pTrc99B and E. coli strain MT102 were gifts from Dr. J. Gangloff of the Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.

Expression and purification of Mm LeuRS, Ec LeuRS and their mutants

The definition of the LSD in Ec LeuRS was based on the crystal structure of Ec LeuRS (PDB entry 4ARC) and sequence alignment. The LSD of Ec LeuRS spans from A 571 to M 617 . Each of the enzymes was expressed in E. coli BL21 (DE3) with a His 6 -tag fused at the N-terminus. The enzymes were purified by affinity chromatography using Ni-NTA (Ni 2+ nitrilotriacetate) Superflow resin, followed by gel-filtration chromatography with Superdex TM 75. The final concentration was determined using a Bradford protein assay as described in the manufacturer’s protocol (Bio-Rad, Hercules, CA, USA).

The genes encoding the various mutants were constructed using the KOD Plus Mutagenesis Kit (Toyobo Life Science) and confirmed by DNA sequencing (BioSun Bioscience). Insertion of the CP1 domain of Ec LeuRS into Mm LeuRS was performed as described previously ( 17 ). Insertion of the LSD of Ec LeuRS into Mm LeuRS-CP1 was performed in several steps. First, the 398 GKDG 401 peptide was deleted from the Mm LeuRS-CP1, and then the 47 amino acid residues from the LSD of the Ec LeuRS (from A 571 to M 617 ) were added progressively by five rounds of mutagenesis.

Preparation of RNA substrates

E. coli tRNA LeuGAG ( Ec tRNA Leu ) with an accepting activity of 1400 pM/A 260 was prepared from overproducing strains constructed in our laboratory ( 18 ). In vitro transcription of Mm tRNA Leu and mutated derivatives was performed as described previously ( 17 ). The accepting activities of the Mm tRNA LeuUAA and Mm tRNA LeuUAG transcripts and the mutated derivatives (A6G, C20U, C67U, V-arm-4 nt, V-arm-5 nt, C20U + V-arm-5 nt, A6G + V-arm-5 nt) were all between 1200–1500 pM/A 260 . [ 3 H]Ile- Ec tRNA Leu , [ 3 H]Ile- Mm tRNA Leu and its mutants were obtained using the editing-deficient Ec LeuRS-Y330D mutant as described previously ( 19 ).

tRNA charging, misacylation and deacylation

Aminoacylation activities of Mm LeuRS, Ec LeuRS and their mutants were measured in a reaction mixture containing 100 mM Tris-HCl (pH 7.8), 30 mM KCl, 12 mM MgCl 2 , 0.5 mM DTT, 4 mM ATP, 10 μM tRNA Leu , 40 μM [ 3 H]Leu (11 Ci/mM) and the enzyme (5 nM Ec LeuRS or 20 nM Mm LeuRS and their mutants). Reactions were carried out at 30°C for Mm LeuRS and the mutants, while Ec LeuRS and derivatives were assayed at 37°C. For _K_m determinations, tRNA concentrations ranged 0.5–30 µM. Misacylation assays were performed under similar conditions, except that 40 µM [ 3 H]Ile (30 Ci/mM; PerkinElmer) and 1 µM of enzyme were used. The deacylation reaction was measured by determining hydrolytic rates, and this was performed at 30°C in 100 mM Tris-HCl (pH 7.5), 30 mM KCl, 12 mM MgCl 2 , 0.5 mM MgCl 2 , 0.5 mM DTT and 1 μM [ 3 H]Ile-tRNA Leu . Reactions were initiated with enzyme diluted to 20 nM. Because radioactive Nva is commercially unavailable, [ 3 H]Ile was used as a source to prepare mischarged tRNA Leu .

AMP formation

The net effect of the editing reaction is the consumption of ATP. Therefore, editing can be measured through AMP formation in the presence of a non-cognate amino acid. AMP formation rates of Mm LeuRS, Ec LeuRS and their mutants were measured as described previously ( 19 ). The reaction mixture contained 100 mM Tris-HCl (pH 7.8), 30 mM KCl, 12 mM MgCl 2 , 5 mM DTT, 5 U/ml pyrophosphatase (Roche), 3 mM ATP, 20 nM [α- 32 P] ATP (3000 Ci/mM; PerkinElmer), 15 mM Nva and the presence or absence of 5 μM tRNA Leu . The reaction was initiated by the addition of 1 μM Mm LeuRS-CP1/LSD (at 30°C), or 0.2 μM for Ec LeuRS and the mutant enzymes (at 37°C). At regular time intervals, aliquots of 1.5 µl were quenched in 6 µl of 200 mM sodium acetate (pH 5.0). Quenched aliquots (1.5 µl each) were spotted in duplicate on polyethyleneimine cellulose plates (PEI, Merck) that had been pre-washed with water. Separation of [ 32 P] aminoacyl-adenylate, [ 32 P]AMP and [ 32 P]ATP was performed by developing TLC plates in the presence of 0.1 M ammonium acetate and 5% acetic acid. Plates were visualized by phosphorimaging, and data were analyzed using Multi Gauge V3.0 software (Fujifilm). The grey densities of [ 32 P]AMP spots were compared with those of known [ 32 P]ATP concentrations. Rate constants ( _k_obs ) were obtained from graphs of [ 32 P]AMP formation plotted against time.

RESULTS

The LSD is not essential for aminoacylation activity and post-transfer editing

Mm LeuRS is an exceptionally small LeuRS that lacks both the CP1 domain and the LSD, and sequence alignment shows that these two domains are replaced by a nonapeptide linker 227 KEEIDGKIT 235 and a tetrapeptide linker 398 GKDG 401 , respectively ( Figure 1 B). Previous studies have shown that the nonapeptide linker from Mm LeuRS can replace the CP1 domain of Ec LeuRS to permit aminoacylation ( 17 ). In this present study, we examined whether the tetrapeptide GKDG from Mm LeuRS could replace the LSD of the Ec LeuRS. The resulting mutant that lacked the LSD was called Ec LeuRS-GKDG. The catalytic efficiency ( _k_cat / _K_m ) of Ec LeuRS-GKDG for Ec tRNA Leu aminoacylation was just more than half of that of the native Ec LeuRS ( Figure 1 C, Table 1 ), indicating that the GKDG sequence of Mm LeuRS could functionally replace the 47 amino acid residues of the LSD in Ec LeuRS. In parallel, we constructed a similar mutant to contain a tetra Ala peptide instead of the GKDG insertion but the resulting mutant ( Ec LeuRS-AAAA) was inactive in the aminoacylation reaction ( Figure 1 C) despite intact folding as shown by CD-spectroscopy analysis ( Supplementary Figure S1 ). Compared with results obtained in a previous study ( 8 ), the GKDG insertion led to much better recovery of activity in Ec LeuRS (55% aminoacylation activity of the wild-type enzyme, Table 1 ). However, the Ec LeuRS-AAAA mutant only displayed 0.55% of the aminoacylation activity of the wild-type enzyme ( Table 1 ), and approximately 1/6 the activity of the previously reported ΔLSD-valRStt mutant (3.5%), which was obtained by using a seven-residue sequence (VLDEKGQ) from T. thermophilus ValRS instead of the LSD of Ec LeuRS ( 8 ). These results indicate that the 398 GKDG 401 of Mm LeuRS is a kind of minimal functional domain. In addition, both Ec LeuRS-GKDG and Ec LeuRS-AAAA exhibited intact deacylation activity for mischarged Ile-tRNA Leu ( Figure 1 D), further proving that the native LSD does not play a critical role during the deacylation of tRNA ( 17 ).

Table 1.

Kinetic constants of various LeuRSs determined in the aminoacylation reaction

D is the discrimination factor of the different LeuRSs for the two bacterial tRNAs, and this value was calculated as follows: D = _k_cat / _K_m ( Ec tRNA LeuGAG ) / _k_cat / _K_m ( Mm tRNA LeuUAG ). Kinetic constants were determined using the tRNA charging assay described in the experimental section except the concentration was 100 nM for Ec LeuRS-AAAA and from 0.2 to 20 μM for tRNAs. All parameters represent the average of three trials with the standard deviations indicated.

a Data from Tan et al. ( 17 ).

Table 1.

Kinetic constants of various LeuRSs determined in the aminoacylation reaction

D is the discrimination factor of the different LeuRSs for the two bacterial tRNAs, and this value was calculated as follows: D = _k_cat / _K_m ( Ec tRNA LeuGAG ) / _k_cat / _K_m ( Mm tRNA LeuUAG ). Kinetic constants were determined using the tRNA charging assay described in the experimental section except the concentration was 100 nM for Ec LeuRS-AAAA and from 0.2 to 20 μM for tRNAs. All parameters represent the average of three trials with the standard deviations indicated.

a Data from Tan et al. ( 17 ).

A mutagenesis study was carried out to further explore the role in Mm LeuRS of the residues of the GKDG peptide. Each of the residues in the tetrapeptide was mutated to Ala separately. All the mutants displayed altered tRNA-charging activity. Moreover, substitution of the flexible Gly398 and Gly401 to rigid Pro residues severely impaired aminoacylation activity to levels comparable with a full deletion of the tetrapeptide linker ( Supplementary Table S1 ). These data suggest that the GKDG peptide of Mm LeuRS plays a critical role in providing flexibility to the catalytic site.

As the two catalytic activities of Ec LeuRS (aminoacylation and editing) do not require the presence of the 47-amino acid LSD, this raises questions concerning the conservation of this module in most prokaryotic LeuRS during evolution.

The LSD of Ec LeuRS favours aminoacylation but inhibits tRNA-independent pre-transfer editing when inserted into Mm LeuRS-CP1

In the next experiments, a series of insertion mutants was constructed to mimic a possible evolutionary process. Chimeric proteins were constructed based on the Mm LeuRS scaffold. First, the LSD of Ec LeuRS was inserted in place of the tetrapeptide GKDG in the Mm LeuRS ( Mm LeuRS-LSD). The resulting Mm LeuRS-LSD mutant did not exhibit any detectable aminoacylation activity (data not shown). Mm LeuRS-CP1 was constructed by inserting the CP1 domain of Ec LeuRS into Mm LeuRS, and this chimeric enzyme had both aminoacylation and editing activities ( 17 ). When Mm LeuRS-CP1 was used as a scaffold to fuse the LSD of Ec LeuRS into its catalytic core, the resulting chimera ( Mm LeuRS-CP1/LSD) had comparable aminoacylation activity to the native Mm LeuRS but demonstrated better catalytic efficiency due to greater affinity with tRNA as indicated by a decrease in _K_m ( Table 1 ). However, the LSD insertion severely decreased the tRNA-independent pre-transfer editing of Mm LeuRS and Mm LeuRS-CP1, and the observed rate constant for AMP formation in the presence of Nva (an analogue of Leu) dropped from 0.16 and 0.12 to 0.037 s −1 ( Table 2 ). In the presence of Ec tRNA Leu and Nva, the observed rate constant of Mm LeuRS-CP1/LSD for AMP formation was comparable with that of Mm LeuRS-CP1, and the rate was 3.6-fold (0.61 s −1 ) greater than that of Mm LeuRS (0.17 s −1 ). This shows that the tRNA-dependent editing pathway became the main editing pathway of Mm LeuRS-CP1/LSD, contributing to 94% of the total editing activity [(0.61 − 0.037)/0.61], whereas the corresponding value in Mm LeuRS-CP1 was just 78% [(0.55 − 0.12)/0.55] ( Figure 2 C). On the other hand, when the LSD of Ec LeuRS was replaced by the GKDG tetrapeptide of Mm LeuRS to form Ec LeuRS-GKDG, the observed rate constant for AMP formation in the presence of Nva of the mutant was 0.75 s −1 compared with 0.33 s −1 for the native Ec LeuRS ( Table 2 ), indicating that the tRNA-independent pre-transfer editing of Ec LeuRS-GKDG contributed much more to total editing (28%; 0.75/2.69) than that of Ec LeuRS (9.6%; 0.33/3.42).

Figure 2.

Effect of LSD mutations on tRNA-independent pre-transfer editing. ( A ) Total editing activity was measured using the AMP formation assay with 0.2 µM Ec LeuRS-GKDG in the absence or presence of 5 µM Ec tRNA Leu and 15 mM Nva. ( B ) A similar assay was performed with 1 µM Mm LeuRS-CP1/LSD in the absence or presence of 5 µM Ec tRNA Leu and 15 mM Nva. ( C ) Contributions of the different editing pathways for each protein: left, sum of the _k_obs of different editing pathways; right, relative contributions of each pathway. Percentages were calculated from _k_obs values of AMP formation reported in Table 1 . tRNA-independent pre-transfer editing was measured in the absence of tRNA. tRNA-dependent editing was deduced by subtracting the tRNA-independent pre-transfer editing from total editing.

Table 2.

Observed rate constants for AMP synthesis in the presence of Nva

All rates represent the average of three trials with the standard deviations indicated.

a Data from Tan et al. ( 17 ).

Table 2.

Observed rate constants for AMP synthesis in the presence of Nva

All rates represent the average of three trials with the standard deviations indicated.

a Data from Tan et al. ( 17 ).

Taken together, these results show that LSD recruiting restricted internal tRNA-independent pre-transfer editing by the synthetic domain of LeuRS. As a consequence, the evolved LeuRS favoured tRNA-dependent pre-transfer editing, which was more effective in maintaining the catalytic fidelity. We propose that this is a possible reason why most prokaryotic LeuRSs have recruited and preserved LSD during their evolution.

The LSD is responsible for tRNA discrimination

In a previous study, it was found that Mm LeuRS-CP1 cross-leucylates Ec tRNA LeuGAG with efficiency comparable with that of the in vitro transcript of Mm tRNA LeuUAG ( 17 ). The present work showed that Mm LeuRS-CP1/LSD aminoacylates more efficiently Ec tRNA Leu than Mm tRNA LeuUAG with a discrimination factor (D factor) of 3.7 (according to _k_cat / _K_m ) ( Table 1 ). Therefore, Mm LeuRS-CP1/LSD had similar discriminatory properties as the native Ec LeuRS, which has a D factor of 5.5 ( Table 1 ). These results suggest that the LSD may participate in tRNA binding and discrimination in some way. When the LSD of Ec LeuRS was replaced by the tetrapeptide GKDG, the mutant Ec LeuRS-GKDG leucylated Ec tRNA LeuGAG and Mm tRNA LeuUAG with similar catalytic efficiency ( Table 1 ). The editing activity of Ec LeuRS-GKDG was also comparable in the presence of Mm tRNA LeuUAG or Ec tRNA LeuGAG ( Table 2 , Supplementary Figure S2 ).

Furthermore, when the CP1 domain and LSD of Ec LeuRS were inserted into Mm LeuRS, the mutant Mm LeuRS-CP1/LSD favoured Ec tRNA LeuGAG not only in aminoacylation but also in editing. In the TLC-based AMP formation assay, Mm LeuRS-CP1/LSD had a rate constant for AMP formation in the presence of Mm tRNA LeuUAG and Nva of 0.21 s −1 , while it was 0.61 s −1 and 0.56 s −1 for Ec tRNA LeuGAG and Mm tRNA LeuUAA , respectively ( Supplementary Figure S2 , Tables 2 and 3 ). Consistently, Ec LeuRS also preferred Ec tRNA Leu in the editing with AMP formation rate of 3.4 s −1 , with a corresponding value of 2.2 s −1 . However, LSD-deprived Ec LeuRS-GKDG showed no preference towards these two tRNAs during editing ( _k_obs 2.7 vs 2.9 s −1 ) ( Table 2 ). These results show that the LSD could confer tRNA discrimination properties to LeuRS, and this raises questions about how the LSD can distinguish tRNAs during aminoacylation and editing.

Table 3.

Kinetic constants of Mm LeuRS-CP1/LSD for mutants of Mm tRNA LeuUAG determined in the aminoacylation reaction

Kinetic constants were determined using the tRNA charging assay described in the experimental section. The concentration of the tRNA Leu s ranged from 0.5–30 μM. All parameters represent the average of three trials with the standard deviations indicated.

Table 3.

Kinetic constants of Mm LeuRS-CP1/LSD for mutants of Mm tRNA LeuUAG determined in the aminoacylation reaction

Kinetic constants were determined using the tRNA charging assay described in the experimental section. The concentration of the tRNA Leu s ranged from 0.5–30 μM. All parameters represent the average of three trials with the standard deviations indicated.

Identification of the critical nucleotides recognized by LSD

To identify the structural determinants of tRNA Leu responsible for LeuRS ability to discriminate tRNA Leu s from various species, we compared the tRNA Leu sequences from E. coli and M. mobile and focused our attention on three differences between them: (i) the sixth base-pair in the acceptor stem of Mm tRNA LeuUAG is a wobble base pair (A 6••• C 67 ), whereas it is a Watson Crick base pair G 6 –C 67 in Ec tRNA LeuGAG ; (ii) the loop of the V-arm of Mm tRNA LeuUAG contains three nucleotides; however, Ec tRNA LeuGAG has a 4-nucleotide loop; (iii) nucleotide 20, located in the ‘variable pocket’ ( 20 ) of the D-loop, is always a U in Ec tRNA Leu s but always a C in Mm tRNA LeuUAG ( Figure 3 A). Therefore, a series of mutants of Mm tRNA LeuUAG was constructed. Firstly, the A 6••• C 67 pair was mutated to a Watson Crick base pair by introducing A6G or C67U mutations. Secondly, in the ‘variable pocket’, nucleotide C20 was changed to a U. Thirdly, the loop of the V-arm was enlarged from three nucleotides to four (V-arm-4 nt) or five (V-arm-5 nt), which are usual sizes for these loops in tRNA Leu s. Mm LeuRS-CP1/LSD leucylated the C20U and V-arm-5 nt mutants at more than twice the catalytic efficiency of the wild-type Mm tRNA LeuUAG (from 0.3 to 0.75 and 0.70 s −1 µM −1 , respectively). Mm LeuRS-CP1/LSD leucylated the double mutant (C20U + V-arm-5 nt), where the C20U mutation and V-arm-5 nt mutation were present, and catalytic efficiency ( _k_cat / _K_m 1.1 s −1 µM −1 ) ( Table 3 ) almost reached the level of Ec tRNA LeuGAG (1.1 s −1 µM −1 in Table 1 ). Similarly, Mm LeuRS-CP1/LSD charged another double mutant (A6G + V-arm-5 nt) and Mm tRNA LeuUAA (another Mm tRNA Leu isoacceptor) with the same catalytic efficiency (0.78 s −1 µM −1 ) ( Table 3 ). Interestingly, we found that Mm tRNA LeuUAA naturally exhibits a large loop of 5 nucleotides in the V-arm according to the genomic tRNA database.

Figure 3.

Mutations in Mm tRNA LeuUAG that impact editing activity. ( A ) Cloverleaf structure of Mm tRNA LeuUAG showing the mutations tested during the study. ( B ) AMP formation assay in the presence of 15 mM Nva catalyzed by 1 µM Mm LeuRS-CP1/LSD in the presence of 5 µM wild-type Mm tRNA LeuUAG , C20U and C20U + V-arm-5 nt. ( C ) Graphical representations of AMP formation as a function of time. _k_obs values of AMP formation were calculated from the slopes, and these are shown in Table 4 .

In the editing reaction, the seven Mm tRNA Leu mutants showed various capacities to stimulate AMP formation. In the presence of Nva, Mm LeuRS-CP1/LSD had a rate constant for AMP formation of 0.21 s −1 for wild-type Mm tRNA LeuUAG ; however, for Mm tRNA Leu -C20U that was increased to 0.41 s −1 . In addition, the most efficient mutant leucylated by Mm LeuRS-CP1/LSD, Mm tRNA Leu -(C20U + V-arm-5 nt), showed very similar effects on editing activity as Mm tRNA LeuUAA in the presence of Nva ( Figure 3 B and C, Supplementary Figure S3 , Table 4 ).

Table 4.

Observed rate constants for AMP synthesis of Mm LeuRS-CP1/LSD in the presence of Nva

All rates represent the average of three trials with the standard deviations indicated.

Table 4.

Observed rate constants for AMP synthesis of Mm LeuRS-CP1/LSD in the presence of Nva

All rates represent the average of three trials with the standard deviations indicated.

A key Lys residue of the LSD is responsible for tRNA discriminatory activity

It has been reported that Ec LeuRS contacts bases 10 and 27 of tRNA Leu via the Arg595 and Arg600 residues located on the so-called β-hairpin of the LSD ( 4 ). To investigate whether these residues could be responsible of discrimination between Ec tRNA LeuGAG and Mm tRNA LeuUAG , initially we mutated the Arg595 and Arg600 of Ec LeuRS to Ala residues. Both mutants, Ec LeuRS-R595A and Ec LeuRS-R600A, showed high catalytic efficiency preference for Ec tRNA LeuGAG but neither reached the value of the wild-type Ec LeuRS ( Table 5 ). However, another mutant on the β-hairpin, Ec LeuRS-K598A, exhibited a stronger effect on aminoacylation activity. Ec LeuRS-K598A displayed a considerably lower affinity for Ec tRNA LeuGAG compared with wild-type Ec LeuRS ( _K_m increased about 4-fold), which resulted in a decrease of the catalytic efficiency by almost 3-fold from 2.2 to 0.84 s −1 µM −1 . On the other hand, Ec LeuRS-K598A bound more tightly with Mm tRNA LeuUAG , and this induced a significant increase in the catalytic efficiency for the leucylation of Mm tRNA LeuUAG (1.1 s −1 µM −1 ) compared with wild-type Ec LeuRS (0.4 s −1 µM −1 ) ( Table 5 ).

Table 5.

Kinetic constants of Ec LeuRS and its mutants in the aminoacylation reaction

D is the discrimination factor of the different LeuRSs for the two bacterial tRNAs, and this value was calculated as follows: D = _k_cat / _K_m ( Ec tRNA LeuGAG ) / _k_cat / _K_m ( Mm tRNA LeuUAG ). All parameters represent the average of three trials with the standard deviations indicated.

a Data from Tan et al. ( 17 ).

Table 5.

Kinetic constants of Ec LeuRS and its mutants in the aminoacylation reaction

D is the discrimination factor of the different LeuRSs for the two bacterial tRNAs, and this value was calculated as follows: D = _k_cat / _K_m ( Ec tRNA LeuGAG ) / _k_cat / _K_m ( Mm tRNA LeuUAG ). All parameters represent the average of three trials with the standard deviations indicated.

a Data from Tan et al. ( 17 ).

In the same way, when K598 in the LSD of chimeric Mm LeuRS-CP1/LSD was replaced with an Ala residue, the catalytic efficiency of the mutant for Mm tRNA LeuUAG was greater than that for Ec tRNA LeuGAG ; however, the catalytic efficiency of Mm LeuRS-CP1/LSD for Mm tRNA LeuUAG was lower than that for Ec tRNA LeuGAG , indicating that mutant Mm LeuRS-CP1/LSD-K598A prefers to charge Mm tRNA LeuUAG , while Mm LeuRS-CP1/LSD prefers Ec tRNA LeuGAG . Thus, mutation at K598 changed the species preference of these enzymes for their tRNA Leu substrates ( Table 5 ). The results show that residue K598 in the β-hairpin of the LSD of Ec LeuRS contributes positively to the binding and aminoacylation of Ec tRNA LeuGAG and acts as an antideterminant versus Mm tRNA LeuUAG . When K598 was mutated to an Ala residue, the antideterminant effect was suppressed and the specific recognition of Ec LeuRS LSD for Ec tRNA Leu was extended to Mm tRNA Leu . In parallel, there was a decrease of binding affinity of Mm LeuRS-CP1/LSD-K598A for Ec tRNA Leu , which reduced its catalytic efficiency to a lower level ( _k_cat / _K_m 0.54 s −1 µM −1 ) than for Mm tRNA LeuUAG ( _k_cat / _K_m 1.4 s −1 µM −1 ). These data show that K598 is a key residue that controls the cross-recognition of tRNA Leu s from different species.

Consistently, in the chimeric Mm LeuRS-CP1/LSD, the K598A mutation controlled post-transfer editing, as there was a drop in the AMP synthesis rate ( Figure 4 A, Table 2 ) and an absence of deacylation activity towards Ile- Ec tRNA LeuGAG ( Figure 4 B). The loss of deacylation properties was further confirmed by a loss of aminoacylation specificity as illustrated by the Ile mischarging of Ec tRNA LeuGAG ( Figure 4 C). Both Mm LeuRS-CP1/LSD-K598A and Mm LeuRS were able to mischarge Ile in contrast to Mm LeuRS-CP1/LSD and Mm LeuRS-CP1 that could not catalyze this substrate. Taken together, these results suggested that the crucial K598 residue of LSD mediated the interaction with tRNA and involved in tRNA recognition.

Figure 4.

Editing and mischarging properties of Mm LeuRS-CP1/LSD-K598A. ( A ) Total editing activity was measured by the AMP formation assay with 1 µM of Mm LeuRS-CP1/LSD-K598A and 15 mM Nva in the absence or presence of 5 µM Ec tRNA LeuGAG or Mm tRNA LeuUAG . ( B ) Deacylation of [ 3 H]-Ile- Ec tRNA Leu (1 µM) by 20 nM of Mm LeuRS (black circle), Mm LeuRS-CP1/LSD-K598A (inverted black triangle), Mm LeuRS-CP1/LSD (black square) and Mm LeuRS-CP1 (black triangle). ( C ) Mischarging of Ec tRNA LeuGAG (20 µM) with Ile catalyzed by 1 µM of Mm LeuRS (black circle), Mm LeuRS-CP1/LSD-K598A (inverted black triangle), Mm LeuRS-CP1/LSD (black square) and Mm LeuRS-CP1(black triangle). ( D ) Crystal structure of tRNA Leu (light blue in the cartoon model) in complex with Ec LeuRS (grey) during the editing conformation (PDB ID code 4ARC, Ref.4). Residues R595, K598 and R600 of LSD (green) are numbered and shown in stick representation with labelling. Both G10 and G46 of tRNA Leu were also highlighted with the stick model with their distances to K598 labelled.

In three of the four crystallographic structures that describe the aminoacylation and proofreading states of LeuRS ( 4 ), the ε-amino group of K598 is located in the vicinity of the phosphate group of G10. K598 approaches the tRNA bound in the editing conformation at distances from 4.6 to 4.9 Å according to the different tertiary structures (in 4ASI, 4ARC and 4ARI). In addition, in the editing complex bound with leucine (4ARC), the ε-amino group of K598 forms a potential interaction with the phosphate group of G46 at a distance of 3.9 Å ( Figure 4 D). However, these putative interactions with the phosphate backbone of tRNA can hardly explain the new discriminating properties of the K598A mutant for Mm tRNA Leu and Ec tRNA Leu . Nevertheless, we cannot exclude the possibility that they play a role during the transition of the 3′ end of tRNA between the aminoacylation and the editing states, and thus favour the aminoacylation of one isoacceptor.

DISCUSSION

With genome sizes <1 Mb, bacteria from the genus Mycoplasma have been described as the ‘smallest free-living organisms’, and thus are considered to be the best representatives for the concept of a minimal cell. The M. mobile genome encodes only 635 proteins ( 21 ), and includes 27 tRNA genes, one of the lowest abundances reported for any organism. Strong evidence suggests that mycoplasmas evolved by a process of reductive evolution that was made possible by adopting a parasitic lifestyle. During this process, the mycoplasmas lost considerable portions of their ancestral chromosomes but retained the genes essential for life. Genome compaction in mycoplasmas is often reflected by the presence of reduced intergenic spacers and by the shortness of most putative proteins relative to their orthologues ( 22 ). aaRSs genes did not escape this size reduction, and several of these enzymes have lost key residues in their editing domains, and in the extreme case of LeuRS, the CP1-editing domain has been deleted completely ( 10 , 17 ). Therefore, mycoplasmas are following a kind of reverse evolution that consists of selecting minimalist proteins that mimic the primitive proteins. Primitive aaRSs have followed an opposite evolutionary pathway by progressively adding domains to improve efficiency and fidelity and to conserve the genetic code and proteome in its present form.

LeuRSs from various species are very complex enzymes that are amongst the largest aaRSs. These enzymes have an unusually high number of modules appended to the catalytic core that participate in a concerted way in tRNA binding, aminoacylation and proofreading. Recent X-ray analysis of tRNA Leu –LeuRS complexes in the aminoacylation or editing conformation has provided the structural basis and dynamics of the aminoacylation and proofreading functional cycle ( 4 ). LeuRS produces error-free Leu-tRNA Leu by coordinating the translocation of the CCA-end of mischarged tRNAs from its synthetic site to the separate proofreading site where the editing occurs. Such translocation involves correlated rotations of four LeuRS domains that are linked to the catalytic core. These motions drive the CCA sequence of the tRNA from the aminoacylation site to the editing site. During this process, the CP1-editing domain stabilizes the tRNA during aminoacylation, while a large rotation of the LSD positions the conserved KMSKS loop of the LeuRS to bind the CCA end of the tRNA, thereby promoting catalysis ( 4 ).

The absence of both CP1 and LSD in Mm LeuRS offers the opportunity to investigate the mechanism of insertion of these additional modules and explore the plasticity of the catalytic core to acquire new functions. Previously, it was shown that insertion of the CP1 domain into the minimal Mm LeuRS did not change synthetic efficiency ( 17 ). CP1 insertion does improve affinity for the tRNA but it decreases _k_cat , suggesting that the tighter binding of the substrate is deleterious for its subsequent reactivity or release. The fusion of the domains of Ec LeuRS with Mm LeuRS also provided the post-transfer editing function to the chimeric enzyme Mm LeuRS-CP1, and this enzyme demonstrated greater activity for E. coli tRNA Leu . Although the post-transfer editing activity of Mm LeuRS-CP1 remained modest compared with that of Ec LeuRS, this observation supports the theory that the aaRS evolved by fusion with additional modules ( 23 ).

Here, we showed that insertion of the LSD of Ec LeuRS into the pre-existing chimeric protein Mm LeuRS-CP1 further improved tRNA binding, leading to a protein with greater catalytic efficiency. In contrast, the editing activity of the double insertion mutant was increased only rather poorly, and a decrease of the pre-existing pre-transfer editing activity of Mm LeuRS was observed. Therefore, fusion with the second insertion domain improved not only tRNA binding and the synthetic activity of the enzyme but it also conferred greater importance to post-transfer editing relative to pre-transfer editing. This change might be explained by adenylate molecules reacting faster with tRNA to synthesize aminoacyl-tRNAs, thereby reducing their opportunity to be edited by the pre-transfer editing process in the synthetic site.

These data provide evidence that the CP1 domain and LSD cooperate for greater synthetic and proofreading properties when inserted in the Mm LeuRS framework, and these observations suggest how these enzymes could have evolved from primitive aaRSs. In this manner, the editing domain, or another domain, could have been distributed amongst different aaRSs before their fine adjustment to the new substrate through the accumulation of mutations. In this present work, we further simulate evolution and show that single mutation events could significantly improve enzyme activity. For instance, mutations could take place in trans in the genes of the corresponding tRNAs. We showed that a mutation at position 20 of Mm tRNA Leu (C20U) doubled the relative activity of Mm LeuRS-CP1/LSD in the aminoacylation and proofreading compared with the wild-type Mm tRNA Leu ( Tables 3 and 4 ). Residue 20 is located in the ‘variable pocket’, and it is known to be a recognition element in different aminoacylation systems ( 20 , 24–26 ). In LeuRS, the only putative interaction of nucleotide 20 occurs with Lys813 that is located in the C-terminal domain, and this can occur only during the editing state (PDB entry 4ARC). Therefore, modifying a specific interaction of the editing state with a distinct module of the enzyme may have improved both synthetic and editing activities. As these activities contribute to a unique functional cycle, any mutation impacting one step may have repercussions on other activities. A similar improvement of catalytic properties was also observed with a double mutant that contained mutations in the acceptor arm and variable arm (A6G + V-arm-5 nt). Here also restoration of activity may occur through the C-terminal domain of LeuRS, which interacts with several nucleotides of the V-arm. Enlarging the loop might have reorganized tRNA binding and pivoting during the catalytic cycle. In addition, Mm tRNA LeuUAA with the natural 5-nt loop exhibited much greater aminoacylation and editing activities and was endowed with the most codon usage in M . mobile . The second mutation (A6G; located at a 50-Å distance in the acceptor arm) might have amplified the first effect ( 4 ).

Additionally, we showed that the synthetic performance of the chimeric enzyme could be improved in cis by a single mutation in the inserted LSD. We have found that Lys598 is an antideterminant for Mm tRNA Leu , but negative effects could be cancelled by Ala mutation. Therefore, this mutant shows that there are at least two alternative ways to improve the aminoacylation–proofreading functional cycle: one way consists of adapting the enzyme by mutating critical amino acids residues, while the second way consists of adjusting the tRNA structure in keeping with the newly inserted modules and the resulting conformation changes that occur during the catalytic processes.

Altogether, our results support the theory that fusion of additional modules to the ancient catalytic core of aaRSs during evolution introduced new catalytic functions to improve fidelity and catalytic performance ( 27 ). Moreover, this present study shows that the minimalist Mm LeuRS is an ideal platform for further studies to understand the evolution of the aaRSs family through the acquisition of complementary modules.

FUNDING

National Key Basic Research Foundation of China [2012CB911000]; The Natural Science Foundation of China [30930022 and 31130064]; Committee of Science and Technology in Shanghai [12JC1409700]; Visiting Professorship for Senior International Scientists from the Chinese Academy of Sciences [2011T2S10]. Funding for open access charge: The Natural Science Foundation of China [30930022 and 31130064].

Conflict of interest statement. None declared.

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Author notes

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.