# Leucyl-tRNA Synthetase Consisting of Two Subunits from Hyperthermophilic Bacteria Aquifex aeolicus

Min-Gang Xu, Jian-Feng Chen, Franck Martin, Ming-Wei Zhao, Gilbert Eriani, En-Duo Wang

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Min-Gang Xu, Jian-Feng Chen, Franck Martin, Ming-Wei Zhao, Gilbert Eriani, et al., Leucyl-tRNA Synthetase Consisting of Two Subunits from Hyperthermophilic Bacteria Aquifex aeolicus. Journal of Biological Chemistry, 2002, 277 (44), pp.41590-41596. 10.1074/jbc.M205126200. hal-04693940

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# Leucyl-tRNA Synthetase Consisting of Two Subunits from Hyperthermophilic Bacteria *Aquifex aeolicus*\*

Received for publication, May 24, 2002, and in revised form, July 19, 2002
Published, JBC Papers in Press, August 25, 2002, DOI 10.1074/jbc.M205126200

Min-Gang Xu‡§, Jian-Feng Chen‡§, Franck Martin¶, Ming-Wei Zhao‡, Gilbert Eriani¶, En-Duo Wang‡‖

From the ‡State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China and ¶UPR9002, Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Rene Descartes, F-67084 Strasbourg Cedex, France

In a hyperthermophilic bacterium, *Aquifex aeolicus*, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (α and β), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of *A. aeolicus* as a template, genes encoding the α and β subunits were amplified and cloned in *Escherichia coli*. The α subunit could not be expressed stably *in vivo*, whereas the β subunit was overproduced and purified by a simple procedure. The β subunit was inactive in catalysis but was able to bind tRNA<sup>Leu</sup>. Interestingly, the heterodimer αβ-LeuRS could be overproduced in *E. coli* cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of *A. aeolicus* LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA<sup>Leu</sup> from *A. aeolicus* and *E. coli* were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mM, 7.8, and 70 °C, respectively. Under optimal conditions, *A. aeolicus* αβ-LeuRS is stable up to 65 °C.

The aminoacyl-tRNA synthetases (aaRSs,<fnref n="1" /> EC 6.1.1.1) catalyze the esterification of amino acids to their cognate tRNAs (1-5). They are specific for both amino acid and tRNA substrates to ensure the high fidelity required by translation (6). The alignments of the sequences of aaRSs and tRNAs have been widely used to study their specific interactions. The 20 aaRSs can be divided into two classes of 10 members each on the basis of conserved sequences and characteristic structural motifs (7). Class I contains two signature peptides, HIGH and KMSKS, located in the active site that is built as the characteristic dinucleotide binding fold (Rossmann Fold, βαβαβ). Connective peptides (called CP domains) are often inserted between the different strands of the Rossmann fold (8). The active site of class II aaRSs is built around an antiparallel β

sheet partly closed by helices, which contains three conserved motifs (4).

Except for arginyl-, glutamyl- and glutaminyl-tRNA synthetase (ArgRS, GluRS and GlnRS) (9, 10), aminoacylation of tRNA catalyzed by the other 17 aaRSs is a two-step reaction: (a) activation of amino acids with ATP by formation of aminoacyl adenylate and (b) transfer of the aminoacyl-moiety from the aminoacyl adenylate to the cognate tRNA substrate (11). The activities of these two steps are independent, and substrate tRNA is unnecessary for amino acid activation. The accuracy of aminoacylation depends on both the specific recognition of amino acids during their activation and the pre- or posttransfer editing that corrects errors at either the stage of aminoacyl adenylate formation or at the step of aminoacyl transfer to tRNA (12-14).

Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) belongs to the class I aaRSs and catalyzes leucylation of tRNA<sup>Leu</sup> on the 2'-OH of the ribose of the 3'-terminal adenosine (7). The primary sequences of LeuRSs from over 50 sources are known. LeuRS are large monomeric enzymes (860 amino acids residues for the *Escherichia coli* enzyme), which are homologous to other synthetases from class Ia: isoleucyl-tRNA synthetases (IleRS), and valyl-tRNA synthetases (ValRS), methionyl-tRNA synthetases (MetRS), arginyl-tRNA synthetases, and cysteinyl-tRNA synthetase (CysRS). LeuRSs are more closely related to the large monomeric IleRS and ValRS, which exhibit large CP domains involved in the editing process and several zinc binding domains.

The conservation of the monomeric structure of these enzymes is universal, except for the LeuRS from *Aquifex aeolicus*, which is an αβ heterodimer (15). On the basis of sequence alignments, CP1 of *E. coli* LeuRS extends from residues 126 to 389, and CP2 spans residues 424-582 (16). *A. aeolicus* is one of the most primitive bacteria. With a growth-temperature maximum near 95 °C, *A. aeolicus* is one of the most thermophilic bacteria known. It grows at 85 °C under an H₂/CO₂/O₂ (79.5: 19.5:1.0) atmosphere in a medium containing only inorganic components. Its complete genome was sequenced in 1998 (15). According to the analysis of its genomic DNA and alignment of the gene sequence of other LeuRSs, the *A. aeolicus* LeuRS appears to be separated into two subunits that were named α and β subunit, respectively.

Here, we report the cloning and expression of the two subunits of *A. aeolicus* LeuRS followed by the purification of the αβ-LeuRS and its β subunit alone. We also report the tRNA binding with its cognate tRNA, the kinetic properties, and thermal stability of these molecules. The cross-recognition of LeuRS and tRNA<sup>Leu</sup> from *E. coli* and *A. aeolicus* was studied.

<footnote>\* This work was funded by Natural Science Foundation of China Grant 30170224, the Chinese Academy of Sciences Grant KSCX-2-2-04, and the exchange program between Chinese Academy of Sciences and CNRS, France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>§ Both authors contributed equally to this work.</footnote>

<footnote>‖ To whom correspondence should be addressed: State Key Laboratory of Molecular Biology, Inst. of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Rd., Shanghai 200031, China. Tel.: 0086-21-64374430; Fax: 008621-64338357; E-mail: wed@server.shcnc.ac.cn.</footnote>

<footnote n="1">¹ The abbreviations used are: aaRSs; aminoacyl-tRNA synthetases; CP, connective peptide; HPLC, high pressure liquid chromatography; CD, circular dichroism.</footnote>
## EXPERIMENTAL PROCEDURES

**Materials**—L-leucine, dithiothreitol, ATP, nucleotide triphosphate, 5'-GMP, tetrasodium pyrophosphate, and inorganic pyrophosphatase were purchased from Sigma. [¹⁴C]leucine (300–400mCi/mmol) and ³²P-labeled tetrasodium pyrophosphate were obtained from Amersham Biosciences. GF/C filters were from Whatman Company. Kinase, ligase, and restriction endonucleases were obtained from Sangon Company, Shanghai Branch. *E. coli* LeuRS was purified by chromatography on DEAE-Sepharose CL-6B and Blue-Sephacel from the overproducing *E. coli* strain in our laboratory (17). Its concentration was determined by A₂₅₀ of the enzyme solution; 1.62 mg of protein/ml equals 1 optical density unit at 280 nm. *E. coli* total tRNA containing 50% of tRNALeu₂ (GAG) was isolated from an overproducing strain constructed in our laboratory (18). T7 RNA polymerase was purified from an overproducing strain in our laboratory (19).

**Plasmids**—pSML104 was constructed from pACYC184 and pKK-233-2 (20). It contains the p15A replicon from pACYC184, the strong trc promoter, a multicloning site, two sequences for transcription termination (T1 and T2) of the ribosomal operon rrnB from pKK233-2, and the resistance to chloramphenicol and tetracycline. Plasmid pBCP378 contains the trc promoter, resistance to ampicillin, and an NdeI site at its translation start point without altering the amino acid sequence of the synthesized protein, while ptcR and the lacIq gene confer inducible and controllable expression (21).

**Cloning and Expression of the Gene Encoding A. aeolicus tRNALeu**—
The *A. aeolicus* genome contains five tRNALeu genes. We cloned and expressed its most frequently used isopeptidator, which decodes CUC codons. The tRNALeu gene with anticodon GAG is located from 5988 to 6072 in the *A. aeolicus* genome. Its sequence is GCCGGAGTGGCGG-AACTGGCAGACGCGCCGTCTTGAGGGGACGGTGCCCTTTACGGGCGTGCGGGTTCACCTCCGCCTCCGGCA (anticodon is in bold italic). The cloning of the *A. aeolicus* tRNALeu gene was performed as outlined previously (22). Six complementary and overlapping oligonucleotides encoding the gene and its complementary chain were synthesized chemically by Sangon Company, Shanghai Branch. Oligonucleotides were phosphorylated, hybridized, and ligated between the EcoRI and PstI sites of pTrec99B and then introduced by transformation into *E. coli* strain MT102. Transformants were checked by DNA sequencing, the cells were grown, and the crude tRNA extracts containing the *A. aeolicus* tRNALeu were isolated as previously described (22).

**Transcription and Labeling of tRNA in Vitro**—Plasmids carrying *A. aeolicus* and *E. coli* tRNALeu genes were prepared by procedures described previously (20). T7 transcripts were generated in a reaction mixture containing 40 mM Tris-HCl (pH 8.0), 5 mM dithiothreitol, 10 mM MgCl₂, 2 mM nucleotide triphosphates, 10 mM 5'-GMP, BstNI-digested template DNA (50 µg/ml), 1 unit/ml of inorganic pyrophosphatase, and 2 mg/ml of pure T7 RNA polymerase for 4 h. The transcripts were purified by 20% (W/V) denaturing PAGE and annealed by heating for 2 min in 2 mM MgCl₂ at 75 °C and allowed to slow cool to 30 °C. The accepting activities of *A. aeolicus* and *E. coli* tRNALeu transcripts were 760 and 650 pmol/A₂₅₀, respectively. The tRNALeu transcripts were incubated with calf intestinal phosphatase for 3 h at 37 °C to remove the 5-terminal phosphate groups. This was followed by a phenol:chloroform extraction. Then, the labeling of the T7 transcripts was performed with T₄ polynucleotide kinase for 1 h at 37 °C in the presence of [γ-³²P]ATP. The labeled tRNALeu transcripts were extracted with phenol, precipitated with ethanol and suspended in an appropriate volume of water.

**Cloning the *A. aeolicus* LeuRS α and β Subunit Genes**—The genomic DNA from *A. aeolicus* was a gift from Dr. R Huber (University of Regensburg, Germany). The genes encoding α and β subunits of LeuRS were amplified by PCR. The α subunit gene with NdeI and HindIII site at its ends was inserted into pBCP378 using the same sites. The gene of β subunit with NdeI and SacI sites at its ends was ligated into pBCP378 or pSML104 using the same sites. *E. coli* TG1 was transformed with the recombinant plasmids.

**Purification of *A. aeolicus* αβ-LeuRS and Its β Subunit**—The recombinant vectors pBCP378-lrsa, pBCP378-lrsb were used to overexpress the individual subunits. To overexpress the heterodimeric form αβ-LeuRS, both pBCP378-lrsa and pSML104-lrsb were co-transformed into the *E. coli* TG1 cells. Transformants harboring the recombinant plasmids were grown in 25 ml of LB with 100 µg/ml of ampicillin and 20 µg/ml of chloramphenicol at 37 °C to stationary phase. Cells were harvested by centrifugation, resuspended in 4 ml of disruption buffer (100 mM Tris-HCl, pH 7.5, 10 mM MgCl₂ and 1 mM EDTA), and sonicated for 6 × 20 s at 15 W with a high-intensity ultrasonic processor (375W model). The crude extract was cleared of cellular debris by

centrifugation at 12,000 rpm for 40 min and then analyzed by SDS-PAGE. Clarified extract was heated to 75 °C for 1 h and centrifuged at 12,000 rpm for 40 min.

For purification, 4 liters of cells were treated as described above. The resulting supernatant was loaded onto a (3 × 18 cm) DEAE-Sepharose CL-6B column equilibrated with lysis buffer and eluted with a potassium phosphate gradient from 50 to 500 mM. The active fractions were pooled, dialyzed against 10 mM potassium phosphate buffer, and applied to a (3.5 × 18 cm) Blue-Sepharose CL-6B column. Elution was performed with a linear gradient of potassium phosphate buffer (from 50 to 500 mM). The fractions containing *A. aeolicus* αβ-LeuRS or its β subunit were pooled and concentrated by dialysis against 10 mM potassium phosphate buffer, pH 6.8, containing 50% glycerol.

**Binding Assay of β Subunit of *A. aeolicus* LeuRS with tRNA**—Protein-tRNA interactions were examined by a gel-shift assay. Increasing concentrations of the β subunit were mixed with 0.05–0.1 µM 5'-[³²P]tRNALeu in a 20-µl volume containing 100 mM Tris-HCl buffer, pH 6.8, 30 mM KCl, 12 mM MgCl₂, 0.1 mM EDTA, and 1 unit/µl RNasin at 20 °C for 30 min. A 20% sucrose solution (3 µl) containing tracer dyes was added immediately before loading. The final mixture was then loaded on a 1-mm thick, 8% native polyacrylamide gel in the reaction buffer for 3 h at 100 V at 15 °C. After electrophoresis the gel was dried and exposed.

**Determination of Molecular Mass**—The molecular mass of αβ-LeuRS from *A. aeolicus* was determined by mass spectrometry analysis using an electrospray ionization-Quadrupole Ion Traps-mass spectrometry apparatus. The molecular mass of the β subunit and its complex with tRNALeu was determined by HPLC on a Superdex 200 HR 10/30 column.

**Circular Dichroism (CD) Spectroscopy**—Protein samples at a concentration of 0.6 mg/ml (for *A. aeolicus* αβ-LeuRS) or 0.2 mg/ml (for β subunit) were analyzed on a Jasco J-715 spectropolarimeter with nitrogen purge at room temperature. A 1-mm path-length cuvette was used, and spectra were accumulated over five scans. Estimation of the secondary structure by CD spectrum was calculated according to the method from Yang (23).

**Enzymatic and Kinetic Assay**—ATP-PPᵢ exchange and aminoacylation activities of LeuRS were measured at 37 °C as described (24). The aminoacylation activity was determined at 37 °C in the reaction mixture containing 100 mM Tris-HCl, pH 7.8, 30 mM KCl, 12 mM MgCl₂, 4 mM ATP, 0.5 mM dithiothreitol, 10 µM tRNALeu, and appropriate amounts of [¹⁴C]-labeled amino acids and enzymes. The kinetic constants of enzymes were determined using various concentrations of the relevant substrates. The same assays were carried out at 60 °C. Pre-steady state kinetics was performed with a quenched-flow apparatus according to Ref. 25. The rate of aminoacylation was measured at 40 °C in the following reaction mixture: 100 mM Tris-HCl, pH 7.8, 30 mM KCl, 12 mM MgCl₂, 4 mM ATP, 3.34 µM tRNA, 5.67 µM [¹⁴C]leucine, 0.1 mg/ml bovine serum albumin, 2.5 mM GSH, and 1.66 µM *A. aeolicus* αβ-LeuRS.

**Active Site Titration**—The titration was performed according to method of A. Fersht (26). In the amino acid activation reaction, when the initial concentration of ATP ([ATP]₀) was much higher than that of *A. aeolicus* αβ-LeuRS ([E]₀), the number of moles of aminoacyl adenylate/mole of *A. aeolicus* αβ-LeuRS, that is the number of active sites (n), may be deduced from the following equation: [ATP]ᵢ = [ATP]₀ - n[E]₀ - n[E]₀ k₂f, where [ATP]ᵢ is the concentration of ATP at time t, k₂ is the pseudo-first-order rate constant for the hydrolysis of the aminoacyl adenylate complex. The intercept (I) of the plot of [ATP]ᵢ against t is [ATP]₀ - n[E]₀. The n may be calculated by the equation: n = ([ATP]₀ - I)/[E]₀ (26). The titration was performed as follows. 20 µM [γ-P³²]ATP (20 µCi/ml), 12 mM MgCl₂, 100 mM Tris-HCl (pH 7.8), 1 mM leucine, and 10 u/ml yeast inorganic pyrophosphatase were incubated at 60 °C. Before adding LeuRS, a 15-µl aliquot of the mixture was withdrawn and added into 200 µl of 7% perchloric acid and 8% activated charcoal in water to determine the zero time radioactivity of ATP for calculation of the relationship between [ATP] and cpm of ATP. The reaction was initiated by adding *A. aeolicus* αβ-LeuRS to a concentration of 5 µM ([E]₀). 15-µl aliquots were taken and quenched in various time intervals (t) as described above. The quenched samples were filtered through Whatman GF/C glass fiber filter, washed, and dried. The [γ-P³²]ATP adsorbed on the charcoal was counted to obtain [ATP]ᵢ values. The number of active sites was calculated through above equation.

## RESULTS

**Cloning and Expression of the tRNALeu Gene**—The plasmid containing *A. aeolicus* tRNALeu (GAG) gene was constructed
<figure><img src="image_2.png" /></figure>

FIG. 1. Schematic diagram of the domain structure of *T. thermophilus* LeuRS and *A. aeolicus* LeuRS. The diagram is based on sequence alignments and *T. thermophilus* tridimensional structure. The two halves of the Rossmann-fold and the helix-bundle domain presumably involved in tRNA-anticodon binding are indicated. The editing domain and the leucine-specific domain where the split occurs in the *A. aeolicus* enzyme are boxed. The consensus sequences HIGH and KMSKS sequences are shown, as well as the two zinc binding modules.

and used to transform *E. coli* MT102. In this strain, which overexpresses tRNA<sup>Leu</sup>, the charge of [¹⁴C]leucine on the tRNA reaches 724 pmol/A<sub>260</sub>. As the accepting activity of pure tRNA<sup>Leu</sup> was considered to be 1600 pmol/A<sub>260</sub>, tRNA<sup>Leu</sup> should be about 45% of the total tRNA. This crude overexpressed *A. aeolicus* tRNA<sup>Leu</sup> was used to assay *A. aeolicus* LeuRS activity and binding property. The low content of endogeneous *E. coli* tRNA<sup>Leu</sup> was considered to be negligible compared with the overexpressed *A. aeolicus* tRNA<sup>Leu</sup>.

**Cloning and Expression of the *A. aeolicus* LeuRS *α* and *β* Subunit Genes**—When compared with the sequence of *Thermus thermophilus* LeuRS, it appears that, except for 30 residues at the C-terminal end, the *α* subunit sequence corresponds to the 580 first residues of the *T. thermophilus* LeuRS (Fig. 1). Except for the first 40 residues, the *β* subunit sequence is similar to the peptide fragment 636–878 of *T. thermophilus* LeuRS. Thus, at the junction of the two peptides, two additional domains of about 30 and 40 residues may form some connecting structure between the two subunits (Fig. 1). Theoretically, the molecular masses of *α* and *β* subunits are 74.0 and 33.5 kDa, respectively. The genes encoding *α* and *β* subunits of LeuRS were amplified by PCR. Both DNA fragments were ligated into pBCP378 or pSML104. The resulting recombinant vectors, pBCP378-lrsa, pBCP378-lrsb and PSML104-lrsb were used to transform *E. coli* TG1. After growth of the different transformants and disruption of the cells, a strong signal corresponding to the *β* subunit was detected by SDS-PAGE (lane 5, Fig. 2A). However the *α* subunit was not detected in the crude extract (lane 4, Fig. 2A). The *β* subunit was purified to 85% homogeneity by heating the crude extract at 75 °C for 30 min and centrifugation (lane 6, Fig. 2A).

**Overproduction and Purification of *A. aeolicus* αβ-LeuRS**—To overexpress the heterodimeric form *αβ-LeuRS*, both pBCP378-lrsa and PSML104-lrsb were co-transformed into the *E. coli* cells. In these conditions, the *α* and *β* subunits were detected in the crude extract (lanes 2 and 3, Fig. 2A). *A. aeolicus* *αβ-LeuRS* was purified to about 70% homogeneity after heating at 75 °C for 30 min followed by centrifugation. Thus, *in vivo*, in the presence of the *β* subunit, the *α* subunit was stable and thermostable.

After growth of the transformants in 4 liters of Luria broth medium (16 g of wet cells) about 20 mg of purified *A. aeolicus* *αβ-LeuRS* was obtained as described under “Experimental Procedures.” The purified *A. aeolicus* *αβ-LeuRS* was 95% homogeneous as determined by SDS-PAGE (lane 2, Fig. 2B). *A. aeolicus* LeuRS consisting of *α* and *β* subunits was active, and the molecular mass of the enzyme was 107.5 kDa as measured by mass spectrometry determination. These data demonstrate that *A. aeolicus* LeuRS is a hybrid dimer active in its dimeric form.

**CD Spectra of *A. aeolicus* αβ-LeuRS and its *β* Subunit**—To

<figure><img src="image_3.png" /></figure>

FIG. 2. SDS-PAGE analysis of *A. aeolicus* LeuRS. A, expression of the *α* and *β* subunits of *A. aeolicus* LeuRS. Crude *E. coli* extracts expressing the different subunits of the *A. aeolicus* LeuRS were separated by SDS-PAGE and compared with protein standards (97.4, 66.2, 55.0, 42.7, 40.0, and 31.0 kDa (from top to bottom, lane 1) and to a non-overproducing strain (lane 7, 30 µg of crude extract). Bacterial extracts expressing both subunits were loaded before and after one-step heating (lane 2, 30 µg of crude extract; lane 3, 5 µg of heated extract). The *α* subunit could not be expressed alone (lane 4, 30 µg of crude extract) as opposed to the *β* subunit (lane 5, 30 µg of crude extract and lane 6, 5 µg of heated extract). The arrows indicate the positions of *α* subunit (top) and *β* subunit (low). B, SDS-PAGE analysis of purified *A. aeolicus* LeuRS (lane 2, 5 µg of purified protein) compared with protein standards (lane 1, 66.7, 43, and 31.0 kDa from top to bottom).

understand why the *α* subunit was unstable, the secondary structures of *A. aeolicus* *αβ-LeuRS* and its *β* subunit alone were compared for their CD spectra. The parameters of their secondary structures were estimated, as summarized in Table I (23). The *β* subunit contains more *α* helix and *β* sheet, less *β* turn and random than *A. aeolicus* *αβ-LeuRS*. Thus, the *β* subunit is more structured and probably more rigid than *A. aeolicus* *αβ-LeuRS*, probably due to the association with the *α* subunit, which is probably less structured and less stable than the *β* subunit as suggested by its inability to be overexpressed *in vivo*.

**Binding of *β* Subunit with tRNA<sup>Leu</sup>**—Molecular mass of the *β* subunit was 33.5 kDa as determined by HPLC gel filtration. It means that *β* subunit is a monomer when expressed independently from the *α* subunit. However, the *β* subunit is inactive and cannot bind ATP and leucine as shown by competition assay and gel-shift assay (data not shown). By gel-shift assay we detected the binding of the *β* subunit to the tRNA<sup>Leu</sup> from *A. aeolicus* (Fig. 3A). The binding constant, *K*<sub>d</sub>, calculated by screening the area of the bands was about 20 ± 3 µM, an unusually high value for an aaRS:tRNA interaction. This is in contrast with the *K*<sub>d</sub> value measured for the whole enzyme (0.5 µM, data not shown). In general, the *K*<sub>d</sub> of aaRS for the cognate tRNA is <2 µM, which means that the binding of the *β* subunit to tRNA<sup>Leu</sup> is not very tight. Interestingly, a supershift appears on increasing the concentration of unlabeled tRNA<sup>Leu</sup> during the competition assay (Fig. 3B). Thus, it seems that the *β* subunit may form two kinds of complexes, the upper band corresponding to the *β* subunit with a probable tRNA/enzyme stoichiometry of 2/1. Alternatively, the *β* subunit can bind labeled *E. coli* tRNA<sup>Leu</sup> (data not shown) and tRNA<sup>Arg</sup> (Fig. 3C) with two forms also. The assay of specificity was extended to tDNA encoding *A. aeolicus* tRNA<sup>Leu</sup>, the tDNA competed with the homologous *A. aeolicus* tRNA<sup>Leu</sup>, but the large complex did not appear (Fig. 3D).

To confirm the existence of two tRNA binding sites on the *β* subunit the complex was generated according to the experimental conditions of Fig. 3 and analyzed by gel filtration on HPLC. Two species were separated, with *M*<sub>r</sub> of 63.5 and 93.3 kDa, corresponding to the *β* subunit associated with one or two
<caption>TABLE I
CD estimates of A. aeolicus αβ-LeuRS and its β subunit</caption>

The concentration of A. aeolicus LeuRS and β subunit was 0.6 mg/mL and 0.2 mg/mL, respectively.

<table><thead><tr><th>Protein secondary structure</th><th>αβ-LeuRS</th><th>β Subunit</th></tr></thead><tbody><tr><td>α-Helix</td><td>33.6%</td><td>38.8%</td></tr><tr><td>β-Sheet</td><td>26.7%</td><td>31.7%</td></tr><tr><td>β-Turn</td><td>5.9%</td><td>0.9%</td></tr><tr><td>Random</td><td>33.8%</td><td>28.6%</td></tr></tbody></table>

<figure><img src="image_4.png" /></figure>

FIG. 3. Binding of tRNA<sup>Leu</sup> on the β subunit of A. aeolicus LeuRS. A, the concentration of labeled tRNA<sup>Leu</sup> from A. aeolicus was 0.05 µM. *Lane 1:* without β subunit; *lane 2-6,* the concentration of β subunit was 3, 8, 13.5, 27, and 41 µM, respectively. B, the incubation system included 27 µM β subunit and 0.1 µM labeled tRNA<sup>Leu</sup> from A. aeolicus with increasing concentration of unlabeled A. aeolicus tRNA<sup>Leu</sup>. *Lane 1:* without unlabeled tRNA; *lane 2-5:* the ratio of unlabeled competing tRNA<sup>Leu</sup> to labeled tRNA<sup>Leu</sup> was 2, 10, 20, and 50, respectively. C, the incubation system included 27 µM β subunit and 0.1 µM labeled tRNA<sup>Leu</sup> from A. aeolicus with increasing concentration of unlabeled E. coli tRNA<sup>Arg</sup>; *lane 1:* without unlabeled tRNA<sup>Arg</sup>; *lane 2-5:* the ratio of unlabeled tRNA<sup>Arg</sup> to labeled tRNA<sup>Leu</sup> was 2, 10, 20, and 50, respectively. D, the incubation system included 27 µM β subunit and 0.1 µM labeled tRNA<sup>Leu</sup> from A. aeolicus with increasing concentration of unlabeled tDNA<sup>Leu</sup> (plasmidic DNA encoding tRNA<sup>Leu</sup>); *Lane 1:* without unlabeled tDNA<sup>Leu</sup>; *lane 2-5:* the ratio of unlabeled tDNA<sup>Leu</sup> to labeled tRNA<sup>Leu</sup> was 2, 10, 20, and 50, respectively.

tRNA<sup>Leu</sup> respectively. Thus, the isolated β subunit exhibits two tRNA binding sites, one of them being nonspecific at the level of its tRNA recognition specificity.

**Active Site Titration**—At 60 °C, the plot of [ATP]<sup>t</sup> against t was a straight line (Fig. 4A). The number of active sites was 1.1 ± 0.1 (the value averaged from three independent determinations), which is consistent with that from E. coli LeuRS (27).

**Kinetic Constants of A. aeolicus αβ-LeuRS**—The specific activities and kinetic parameters of A. aeolicus αβ-LeuRS in amino acid activation and aminoacylation reactions were as sayed at 37 °C in order to directly compare them with those of E. coli LeuRS under reaction conditions, which are optimized for that of the E. coli enzyme.

The E. coli and A. aeolicus LeuRS display a remarkable difference in the aminoacylation reaction conditions. For the A. aeolicus enzyme, the magnesium/ATP ratio required for optimal activation and charging reaction is 3 (8 mM excess of free magnesium), an unusually high value for a class I amino- acyl-tRNA synthetase. Compared with the stoichiometric ratio of magnesium/ATP used for the E. coli enzyme (28), this sug- gested that additional magnesium ions are used by the ther- mophilic enzyme to stabilize the productive enzyme-substrate complex. In the ATP-pyrophosphate exchange reaction the Michaelis-Menten constant (K<sub>m</sub>) values for leucine and ATP were 1.3 and 360 µM, respectively, and k<sub>cat</sub> were 3.5 and 3.3

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

FIG. 4. Active site titration and pre-steady state aminoacylation. A, the number of active sites. The active site titration was performed by measuring ATP exhaustion in the formation of leucyl adenylate at 60 °C, pH 7.8, from A. aeolicus αβ-LeuRS (5 µM), [γ-P<sup>32</sup>]ATP (20 µM, 20 µCi/ml) and leucine (1 mM) in the presence of pyrophosphatase. Before adding LeuRS, the number of counts was 19,600 cpm. The ordinate intercept of the extrapolated straight line was 14,300 cpm equivalent to 14.6 µM. The number of active sites was 1.08 calculated by the equation: n = ([ATP]<sub>0</sub> − I)/[E]<sub>0</sub> = (20 − 14.6)/5 = 1.08. B, biphasic tRNA<sup>Leu</sup> aminoacylation kinetic. The reaction was conducted by a quenched flow apparatus. One syringe contained tRNA<sup>Leu</sup>, the other contained the preformed enzyme:adenylate complex.

s<sup>-1</sup>, respectively. Compared with the E. coli LeuRS, these values only reach 2% of the level of activity (Table II, (24)). In the aminoacylation reaction, K<sub>m</sub> values for leucine, ATP, and tRNA<sup>Leu</sup> (A. aeolicus) were 6.4, 550, and 0.38 µM; k<sub>cat</sub> values were 0.39 s<sup>-1</sup> (Table II). Except K<sub>m</sub> for ATP in aminoacylation reaction, all of the constants were lower than those for E. coli LeuRS (Table II) (24). Although the substrates bind A. aeolicus αβ-LeuRS tightly, the reaction is slower at 37 °C.

The kinetic constants of A. aeolicus αβ-LeuRS were also studied at 60 °C (Table III). All k<sub>cat</sub> values increased 3.5–4.8-fold as compared with those at 37 °C. In amino acid activation, the K<sub>m</sub> values for both leucine and ATP were identical to those at 37 °C. In the aminoacylation reaction, the K<sub>m</sub> value for leucine was unchanged, whereas the K<sub>m</sub> value for ATP was slightly increased. At 60 °C, the catalytic efficiencies observed for the different substrates are comparable to those of the E. coli LeuRS at 37 °C.

The aminoacylation reaction was studied at the pre-steady state level using a quenched flow apparatus (25). The reaction was started by mixing the tRNA to the preformed enzyme: adenylate complex. A two-phase kinetics was detected, with an
<caption>TABLE II</caption>

Kinetic constants for LeuRSs from A. aeolicus and E. coli at 37 °C

The kinetic constants of enzymes were determined as described under "Experimental Procedures." All the data in this table were the average values with a variation of <5% from three independent determinations.

<table><thead><tr><th rowspan="2">Substrate</th><th rowspan="2">Constant</th><th colspan="2">ATP-PP<sub>i</sub> exchange</th><th colspan="2">Aminoacylation</th></tr><tr><th>A. aeolicus<br>LeuRS</th><th>E. coli<sup>a</sup><br>LeuRS</th><th>A. aeolicus<br>LeuRS</th><th>E. coli<sup>a</sup><br>LeuRS</th></tr></thead><tbody><tr><td rowspan="3">Leucine</td><td>K<sub>m</sub> (µM)</td><td>1.3</td><td>30</td><td>6.4</td><td>15</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td>3.5</td><td>155</td><td>0.39</td><td>3.0</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td>2692</td><td>5167</td><td>61</td><td>200</td></tr><tr><td rowspan="3">ATP</td><td>K<sub>m</sub> (µM)</td><td>360</td><td>750</td><td>550</td><td>280</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td>3.3</td><td>140</td><td>0.39</td><td>3.6</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td>9.2</td><td>187</td><td>0.71</td><td>12.9</td></tr><tr><td rowspan="3">A. aeolicus<br>tRNA<sup>Leu</sup></td><td>K<sub>m</sub> (µM)</td><td></td><td></td><td>0.38</td><td>1.1</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td></td><td></td><td>0.39</td><td>1.6</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td></td><td></td><td>1026</td><td>1455</td></tr><tr><td rowspan="3">E. coli<br>tRNA<sub>2</sub><sup>Leu</sup></td><td>K<sub>m</sub> (µM)</td><td></td><td></td><td>0.76</td><td>1.5</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td></td><td></td><td>0.084</td><td>2.9</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td></td><td></td><td>111</td><td>1933</td></tr></tbody></table>

<caption>TABLE III</caption>

Kinetic constants for LeuRSs from A. aeolicus at 60 °C

<table><thead><tr><th>Substrate</th><th>Constant</th><th>ATP-PP<sub>i</sub> exchange</th><th>Aminoacylation</th></tr></thead><tbody><tr><td rowspan="3">Leucine</td><td>K<sub>m</sub> (µM)</td><td>1.6</td><td>6.0</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td>15.5</td><td>1.4</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td>9688</td><td>233</td></tr><tr><td rowspan="3">ATP</td><td>K<sub>m</sub> (µM)</td><td>380</td><td>112.3</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td>14.5</td><td>1.8</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td>38.2</td><td>16.0</td></tr><tr><td rowspan="3">A. aeolicus tRNA<sup>Leu</sup></td><td>K<sub>m</sub> (µM)</td><td></td><td>0.3</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td></td><td>1.5</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td></td><td>5000</td></tr><tr><td rowspan="3">E. coli tRNA<sub>2</sub><sup>Leu</sup></td><td>K<sub>m</sub> (µM)</td><td></td><td>1.5</td></tr><tr><td>k<sub>cat</sub> (s<sup>-1</sup>)</td><td></td><td>0.4</td></tr><tr><td>k<sub>cat</sub>/K<sub>m</sub> (s<sup>-1</sup> mM<sup>-1</sup>)</td><td></td><td>267</td></tr></tbody></table>

initial faster rate ($k_{cat}$ = 7.2 s$^{-1}$) followed after 52 ms by a slower rate with a $k_{cat}$ value of 0.36 s$^{-1}$, which is similar to the value measured by hand in steady state, 0.39 s$^{-1}$. The Leu-tRNA$^{Leu}$ synthesized during the burst was in a 0.82:1.0 stoichiometric ratio with the synthetase present (Fig. 4B). Thus, the decrease of the rate of tRNA$^{Leu}$ charging after completion of the first catalytic cycle of the synthetase suggests that one or several end products dissociate slowly from the enzyme.

**Thermal Properties of A. aeolicus αβ-LeuRS**—As expected, A. aeolicus αβ-LeuRS is a thermostable enzyme. Under the experimental conditions it is stable up to 70 °C (Fig. 5). The specific activities of amino acid activation and aminoacylation increased with temperature. At 75 °C the two activities were 12- and 5-fold greater than at 37 °C. Changes of activity with temperature are shown in Fig. 6. The activation energy was calculated from the slope of the line when log v was plotted against 1/T. The activation energy for the amino acid activation reaction was 14.9 kcal/mol. Below 50 °C, the activation energy for the aminoacylation reaction was 14.9 kcal/mol, the same as in amino acid activation. Above 50 °C the value was 6.53 kcal/mol. Above 75 °C, evaporation in the test tube prevented accurate determination of activation energy. The aminoacylation rate began to decrease above 70 °C, 15 °C under its optimum of 85 °C (15). This suggests that the composition of the reaction mixture is not optimal for enzyme activity, or that the A. aeolicus tRNA$^{Leu}$ overproduced in E. coli does not possess all the modified bases that should improve its thermal stability.

**Cross-recognition of LeuRSs and tRNA$^{Leu}$ from A. aeolicus and E. coli**—At 37 °C, both enzymes display for their homologous substrates comparable $k_{cat}/K_m$ values, with the faster rate for the E. coli enzyme and the best tRNA affinity for the A. aeolicus enzyme (Table II). In the cross-recognition of the tRNAs, the Aquifex enzyme exhibits a 5-fold decrease of the $k_{cat}$

<figure><img src="image_7.png" /><figcaption>FIG. 5. Determination of the thermal stability of A. aeolicus αβ-LeuRS. A. aeolicus αβ-LeuRS (40 µg/ml) in 50 mM potassium phosphate buffer, pH 6.8, containing 400 µg/ml bovine serum albumin was incubated at various temperatures for 10 min. Then the aminoacylation activity was assayed at 37 °C after dilution of the reaction mixture with cold 50 mM potassium phosphate buffer, pH 7.5.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>FIG. 6. Variation of the A. aeolicus αβ-LeuRS activity with the temperature. The ATP-PP<sub>i</sub> exchange activity (A) and the aminoacylation reaction (B) were assayed at increasing temperatures.</figcaption></figure>

in the aminoacylation of the E. coli tRNA and a 2-fold decrease of the tRNA affinity. The resulting 10-fold decrease of $k_{cat}/K_m$ suggests that the heterologous Aquifex αβ-LeuRS/ E. coli tRNA$^{Leu}$ complex does not reach an optimal conformation for catalysis. Increasing the temperature increases the $k_{cat}$ to 0.4 s$^{-1}$, still seven times lower than that of E. coli LeuRS at 37 °C.

DISCUSSION

Analysis of the DNA sequence of A. aeolicus LeuRS revealed that this organism presents an unusual split LeuRS in contrast

<footnote><sup>a</sup> The data are from reference 17.</footnote>
to the monomeric form found in all other sequenced LeuRS genes (15). However, despite this difference in quaternary structure, extensive sequence homologies are found with other LeuRSs, with a conservation of the HIGH sequence (HMGH), KMSKS sequence (-MSKS) and CP domain presumably involved in editing. The enzyme is split at the level of the active site, between the two halves of the Rossmann fold, and before the second consensus sequence KMSKS. By analogy with the *T. thermophilus* LeuRS whose structure has been solved by x-ray crystallography, the split should occur in the additional domain preceding the KMSKS sequence (29).

Based on the sequence homologies and x-ray structure of the *T. thermophilus* enzyme, we can postulate that the *β* subunit of the *A. aeolicus* enzyme possesses half of the active site, with the CP domain generally involved in editing attached to the Rossmann fold (Fig. 1) (29). The *β* subunit should contain the second part of the active site and the helix bundle domain found in the class Ia synthetases (Arg-, Met-, Val- and IleRS). This domain is involved in tRNA-anticodon binding as shown by x-ray crystallography (30–32) and functional studies (33, 34). An additional domain located at the C-terminal end of the *α* subunit, characteristic of the *T. thermophilus* and *A. aeolicus* enzymes corresponds to the split site for the *A. aeolicus* enzyme. In light of the structural data, one may imagine that the *β* subunit is stably expressed due to the central helix bundle domain that may strongly structure the subunit. On the other hand, the *α* subunit is unstable apart from the *β* subunit, perhaps due to its modular structure composed of two large domains, namely half of the Rossmann fold and the whole CP domain (200 residues), connected by long hinge peptides, which may induce flexibility and the observed instability of the *A. aeolicus* enzyme (29).

The *β* subunit is small; however, *in vivo* or *in vitro* it is stable and seems to be a chaperon of the *α* subunit in *vivo*. Although inactive in its monomeric form, the *β* subunit binds tRNA<sup>Leu</sup> with low affinity ($K_d$ = 20 ± 3 µM). Compared with the dimeric form αβ ($K_d$ = 0.5 µM, $K_m$ = 0.38 µM), it is a 40-fold difference, which may account for the loss of interactions with the acceptor arm of the tRNA molecule, assuming that the tRNA binds the *β* subunit in its original mode. A second, unspecific tRNA binding site was characterized on the *β* subunit, which may be related to the results observed for ValRS (35). Recently it has been shown that LeuRS from different sources can substitute for the splicing function of yeast mitochondrial LeuRS (36). Thus, LeuRSs intrinsically possess the ability to bind diverse RNA substrates, even larger than tRNA. This may explain why two tRNA molecules can bind simultaneously.

The low affinity value exhibited by the *β* subunit for its tRNA is similar to the values measured for the additional domains found in eukaryotic synthetases, like the C-terminal extension of MetRS related to EMAPII ($K_d$ = 15 µM) (37), the repeated domains of GluProRS ($K_d$ = 2.5–30 µM) (38, 39) and the p43 component of the mammalian multisynthetase complex (40 µM) (40). It has been shown that the additional domains increase the catalytic efficiency of the synthetase without contributing to the specificity. Thus, they can be partially compared with the *β* subunit of the *Aquifex* enzyme, which represents a later stage in the evolution at which point the added domain became more specific for the tRNA substrate.

The αβ-LeuRS from *A. aeolicus* is split in the middle of the bipartite Rossmann fold domain in a place where the ancestor two half Rossmann folds were juxtaposed in order to give the actual class I active site. Thus, the split *A. aeolicus* enzyme seems to have gone back in evolution to a stage when the class I active site was not assembled as the actual bipartite Rossmann fold.

The dimerization of the *A. aeolicus* enzyme raises other

questions concerning the assembly of the subunits. No additional domains involved in assembly are detectable by sequence analysis. Thus, the intramolecular contacts found in the monomeric enzyme should have been transformed into intermolecular contacts after separation of the gene into pieces. Previous work on *E. coli* IleRS demonstrates that this enzyme, closely related with LeuRS, could be expressed in different combinations of split enzymes (16, 41). Thus, it seems that these large monomeric enzymes have the built-in capacity to be synthesized as heterodimers. Similarly, four pairs of fragments corresponding to N- and C-terminal parts of *E. coli* LeuRS with junctions at different positions in the CP domain were expressed and assembled *in vivo*. Except the pair with the junction at the level Glu-292-Ala-293, the assembled proteins were active for aminoacylation (42). However, the Glu-292-Ala-293 mutant still supported the ATP-PPᵢ exchange activity (42). In fact, the assembled *E. coli* LeuRS mimicked a heterodimer LeuRS. We recently tried to split the *E. coli* LeuRS in order to mimic the *α* and *β* subunits of *A. aeolicus* LeuRS; however, the fragments were not stably expressed in *E. coli*.

**Acknowledgments**—We are grateful to Dr. Louis Levinger for carefully reading the manuscript and for helpful suggestions. We warmly thank Dr. Jean Gangloff for advice and involvement in the pre-steady state measurements. We are indebted to Dr. R. Huber for the generous gift of the *A. aeolicus* genomic DNA.

**Addendum**—A recent article (43), published during the preparation of this manuscript, described the heterodimeric structure of LeuRS from *A. aeolicus*. The authors overexpressed separately the subunits of the enzyme and reconstituted *in vitro* the active heterodimeric structure. To obtain the active heterodimer, the *α* subunit was solubilized by urea and renatured in the presence of the *β* subunit. The final mixture was fractionated by gel filtration. The reconstituted heterodimer displayed kinetic constant values close to those we measured except those for tRNA aminoacylation, which were 1–2 orders of magnitude lower. This may take into account the origins of the tRNA substrates. T7 transcripts of the *A. aeolicus* tRNA<sup>CAG</sup> and total *E. coli* tRNA were used instead of overexpressed *A. aeolicus* tRNA<sup>GAG</sup> and purified *E. coli* tRNA<sup>GAG</sup> in our assays.

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