Báo cáo khoa học: Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl–tRNA synthetase and determination of its oligomeric structure in solution pptx

Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl–tRNA synthetase and determination of its oligomeric structure in solution Stefano Paravisi1, Gianluca Fumagalli1, Milena Riva1, Paola Morandi1, Rachele Morosi1, Peter V ´ ˆ Konarev2,3, Maxim V Petoukhov2,3, Stephane Bernier4, Robert Chenevert4, Dmitri I Svergun2,3, Bruno Curti1 and Maria A Vanoni1 ` Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita degli Studi di Milano, Italy European Molecular Biology Laboratory, Hamburg, Germany Institute of Crystallography, Russian Academy of Sciences, Moscow, Russia ´ ´ Department de Chimie, CREFSIP, Universite Laval, Canada Keywords glutamyl–tRNA reductase; glutamyl–tRNA synthetase; Mycobacterium tuberculosis; protein synthesis; tetrapyrrole synthesis Correspondence M A Vanoni, Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita’ degli Studi di Milano, Via Celoria 26, 20131 Milan, Italy Fax: +39 025 031 4895 Tel: +39 025 031 4901 E-mail: maria.vanoni@unimi.it (Received 15 October 2008, revised 23 December 2008, accepted 24 December 2008) doi:10.1111/j.1742-4658.2009.06880.x Mycobacterium tuberculosis glutamyl–tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity It was found to be similar to the other well-characterized GluRS, especially the E coli enzyme, with respect to the requirement for bound tRNAGlu to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters kcat (130 min)1) and KM for tRNA (0.7 lm) and ATP (78 lm), but to differ by a one order of magnitude higher KM value for l-Glu (2.7 mm) At variance with the E coli enzyme, among the several compounds tested as inhibitors, only pyrophosphate and the glutamyl-AMP analog glutamol-AMP were effective, with Ki values in the lm range The observed inhibition patterns are consistent with a random binding of ATP and l-Glu to the enzyme–tRNA complex Mt-GluRS, which is predicted by genome analysis to be of the non-discriminating type, was not toxic when overproduced in E coli cells indicating that it does not catalyse the mischarging of E coli tRNAGln with l-Glu and that GluRS ⁄ tRNAGln recognition is species specific Mt-GluRS was significantly more sensitive than the E coli form to tryptic and chymotryptic limited proteolysis For both enzymes chymotrypsin-sensitive sites were found in the predicted tRNA stem contact domain next to the ATP binding site Mt-GluRS, but not Ec-GluRS, was fully protected from proteolysis by ATP and glutamolAMP Small-angle X-ray scattering showed that, at variance with the E coli enzyme that is strictly monomeric, the Mt-GluRS monomer is present in solution in equilibrium with the homodimer The monomer prevails at low protein concentrations and is stabilized by ATP but not by glutamol-AMP Inspection of small-angle X-ray scattering-based models of Mt-GluRS reveals that both the monomer and the dimer are catalytically Abbreviations aaRS, aminoacyl–tRNA synthetases; ALA, d-amino levulinic acid; ALAS, d-amino levulinic acid synthase; ArgRS, arginyl–tRNA synthetase; Bs-GluRS, Bacillus subtilis glutamyl–tRNA synthetase; D-GluRS, discriminating glutamyl–tRNA synthetase; DLS, dynamic light scattering; E, total enzyme concentration; Ec-GluRS, Escherichia coli glutamyl–tRNA synthetase; GlnRS, glutaminyl–tRNA synthetase; GluRS, glutamyl– tRNA synthetase; GluTR, glutamyl–tRNA reductase; GluTR-His, GluTR carrying a C-terminal His6-tag; GoA, glutamol-AMP; GSA, glutamate 1-semialdehyde; GSA-AM, GSA aminomutase; His6-GluRS, GluRS carrying a N-terminal His6-tag; IPTG, isopropyl thio-b-D-galactoside; LysRS, lysyl–tRNA synthetase; Mt-GluRS, M tuberculosis GluRS; ND-GluRS, nondiscriminating GluRS; PPi, pyrophosphate; SAXS, small angle X-ray scattering; Te-GluRS, Thermosynechococcus elongatus GluRS; Tt-GluRS, Thermus thermophilus GluRS; b-ME, 2-mercaptoethanol 1398 FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al M tuberculosis glutamyl–tRNA synthetase active By using affinity chromatography and His6-tagged forms of either GluRS or glutamyl–tRNA reductase as the bait it was shown that the M tuberculosis proteins can form a complex, which may control the flux of Glu–tRNAGlu toward protein or tetrapyrrole biosynthesis Mycobacterium tuberculosis infects over two-thirds of the world population and causes 1.6 million deaths every year, according to World Health Organization estimates [1] The intrinsic resistance of M tuberculosis to most antibiotics and the spread of multidrugresistant strains prompted the study of M tuberculosis metabolism and the identification of novel antitubercular drug targets through the in vitro characterization of essential enzymes With this goal in mind we focused on the production and characterization of M tuberculosis glutamyl–tRNA synthetase (MtGluRS) Glutamyl–tRNA synthetases (GluRS) belong to the broad class of aminoacyl–tRNA synthetases (aaRS), which catalyse the essential charging reaction of tRNA with the cognate amino acid ensuring correct translation of the mRNA into the corresponding polypeptide [2] The ubiquity and essentiality of aaRS makes them of interest as targets of new anti-infectives [3] Their reaction formally consists of the activation of the amino acid by adenylation (Eqn 1) followed by transfer of the amino acyl residue to the 2¢-OH or 3¢-OH position of the 3¢-OH end of the cognate tRNA (Eqn 2) amino acid ỵ ATP $ aminoacyl-AMP + pyrophosphate ð1Þ amino acyl-AMP + tRNAaa $ AMP + aminoacyl À tRNAaa ð2Þ Most aaRS catalyse the formation of the aminoacylAMP intermediate in the absence of tRNA However, GluRS, glutaminyl–tRNA synthetase (GlnRS), arginyl–tRNA synthetase (ArgRS) and class I lysyl–tRNA synthetase (LysRS) are exceptions in that activation of the amino acid requires the presence of the cognate tRNA [2,4] In these aaRS the binding of tRNA induces an ATP productive binding mode [5] GluRS are also distinguished on the basis of their ability to discriminate between tRNAGlu and tRNAGln The discriminating GluRS (D-GluRS) only catalyses the charging reaction of tRNAGlu with l-Glu yielding Glu–tRNAGlu However, the nondiscriminating GluRS (ND-GluRS) also charges the tRNAGln forming a misacylated Glu–tRNAGln The organisms containing the ND-GluRS also contain a specific Glu–tRNAGln amidotransferase that converts the glutamyl moiety into a glutaminyl residue correcting the misacylation and providing the Gln–tRNAGln needed for protein synthesis [2,6,7] In these cells, the GlnRS is missing Furthermore, in most bacteria and plants GluRS also plays a role in tetrapyrrole biosynthesis, which requires GluRS (Eqn 3), Glu–tRNA reductase (GluTR; Eqn 4) and glutamate 1-semialdehyde aminomutase (GSAAM; Eqn 5) for synthesis of d-aminolevulinic acid (ALA), the first common precursor of all tetrapyrroles [8,9] This C5 pathway of tetrapyrrole biosynthesis differs from that of most eukaryotes and other bacteria, which uses succinyl-CoA, glycine and ALA synthase (ALAS; Eqn 6), the so-called C4 pathway of tetrapyrrole biosynthesis L-Glu + ATP + tRNAGlu $ Glu À tRNAGlu 3ị Glu tRNAGlu + NADPH + Hỵ $ GSA + NADPỵ 4ị GSA $ ALA 5ị succinyl-CoA + glycine $ ALA + CoA ð6Þ How the flux of Glu–tRNAGlu is directed toward protein or tetrapyrrole biosynthesis has not been fully clarified Most likely, different mechanisms operate in different organisms In general, the low levels of GluTR, catalysing the rate-limiting step of ALA biosynthesis, may be sufficient to ensure ALA supply without interfering with protein synthesis [9] However, GluTR may distinguish between different Glu–tRNAGlu isoforms [10] As an alternative, complex formation between GluRS and GluTR, as a function of the cell requests, may divert Glu–tRNAGlu toward tetrapyrrole biosynthesis [11] Finally, GluRS isoforms differing in tRNAGlu specificity [10] or, in principle, in their ability to interact with GluTR may be expressed Only the structures of GluRS from thermophilic bacteria have been solved The Thermus thermophilus enzyme (Tt-GluRS) is the structural model for D-GluRS [5,12–14], and the Thermosynechococcus elongatus form (Te-GluRS) is the structural model for the ND-GluRS class [15] Thus, details of the structure, flexibility, oligomeric state and conformational states of a mesophilic enzyme are not known, limiting to some extent thorough understanding of the FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1399 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al structure–function relationship in this enzyme with consequences for the rational design of specific inhibitors Analysis of the M tuberculosis genome sequence led to the identification of one ORF encoding a putative GluRS (Rv2992c, 1473 bp) Upstream of Rv2992c the sequences of one tRNAGlu (gluU) and one tRNAGln (glnU) gene are found Additional tRNAGlu (gluT) and tRNAGln (glnT) genes have been annotated, but they are in chromosome regions far from the putative GluRS gene and the tetrapyrrole biosynthetic genes (see below) No ORF encoding a putative GlnRS was found, but three ORFs (Rv3011c, Rv3009c and Rv3012c) predict the presence of the three subunits of the Glu–tRNAGln amidotransferase These observations suggest that the Mt-GluRS is of the nondiscriminating type Finally, genes encoding the putative GluTR (hemA, Rv0509), GSA-AM (hemL, Rv0524) and other enzymes of the tetrapyrrole biosynthetic pathway have been annotated in the M tuberculosis genome No ORF encoding proteins similar to ALAS have been found Thus, Mt-GluRS is predicted to provide Glu– tRNAGlu for both protein and tetrapyrrole biosynthesis, the latter occurring via the C5 pathway Furthermore, the genome-wide gene inactivation experiments of Sassetti et al [16] indicate that the putative GluRS, as well as glutamyl–tRNAGln amidotransferase and the enzymes of the C5 pathway of ALA biosynthesis are essential for M tuberculosis For these reasons, with the dual goal of contributing to understanding of the metabolism of this pathogen and providing the enzyme for the identification and development of selective inhibitors, we cloned and expressed Rv2992c in E coli With the purified protein we carried out a kinetic, mechanistic and structural characterization of the resulting Mt-GluRS Rv0509, encoding the putative M tuberculosis GluTR (Mt-GluTR) was also cloned in vectors for protein production in E coli in order to ask questions about GluRS–GluTR complex formation for the mycobacterial proteins Results Expression of Rv2992c in E coli BL21(DE3) and purification of the putative Mt-GluRS The predicted ORF Rv2992c was cloned in pET-based vectors for production of the corresponding protein product in E coli BL21(DE3) The pETGTS1 plasmid coded for a 490-residue protein (53 831 Da), which was produced at high levels and in a soluble form in 1400 E coli (Fig S1A) Similar results were obtained with cells transformed with pETGTS2, which encodes a fusion between an N-terminal His6 tag and the Rv2992c coding region (510 residues and a predicted mass of 55 876 Da; Fig S1B) Up to 100 mg of homogeneous protein, as judged by SDS ⁄ PAGE (Fig S1A), were obtained from 20 g of E coli BL21 (DE3) cells harboring pETGTS1 The His6-tagged variant of the putative GluRS (His6– GluRS) could be purified to homogeneity (Fig S1B) using a single nitrilotriacetic acid–Sepharose column ($ 10 mgỈg)1 of cells) Both protein species could be concentrated to up to 40 mgỈmL)1 without observable precipitation They were stable for up to years when stored at )20 °C in 50% glycerol, as judged by SDS ⁄ PAGE, determination of the protein concentration after centrifugation, activity (see below) and dynamic light scattering (DLS) measurements Glycerol removal by either dialysis or gel filtration led to soluble protein that maintained activity for up to week when stored at °C Freezing samples from which glycerol had been removed caused the aggregation of a small fraction (< 5%) of the protein, as determined by DLS without, however, causing detectable activity loss N-Terminal sequencing and mass determination by MALDI-TOF confirmed the identity of the proteins and that the N-terminal Met residue had been correctly removed by post-translational processing Identification of Rv2992c as the Mt-GluRS and steady-state kinetic characterization Formation of Glu–tRNAGlu was monitored by measuring the increase in acid-precipitable radioactivity upon incubation of the enzyme with E coli tRNAGlu, l-[U14C]Glu, ATP, MgCl2 at pH 7.3 The increase in l-[U14C]Glu–tRNAGlu concentration was linear for up to 10 when 0.2–1.85 pmol Mt-GluRS was used in the reactions (Fig S2) Under these conditions the enzyme had an apparent turnover number of 17.7 ± 0.3 min)1 at 0.5 mm l-Glu and 32.0 ± 1.2 min)1 at mm l-Glu Similar activity was measured with the homogeneous His6-GluRS form (Fig S2) These values are lower than that of $ 100 min)1 calculated from the specific activity reported for the E coli enzyme by Lin et al [17] The activity was found to increase hyperbolically with MgCl2 concentrations up to mm At concentrations > 10 mm the activity decreased Thus, in all assays MgCl2 concentration was held constant at 10 mm, well above ATP concentrations (or its analogs, see below), but below the onset of inhibition FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al M tuberculosis glutamyl–tRNA synthetase Table Steady-state kinetic parameters of the Mt-GluRS reaction The kcat and KM values for ATP, L-Glu and E coli tRNAGlu were determined for the aminoacylation reaction catalysed by Mt-GluRS (6.3 nM) at 37 °C in the presence of 35 mM Hepes ⁄ NaOH buffer, pH 7.3, 25 mM KCl, 10% glycerol, mM dithiothreitol, 10 mM MgCl2 and 0.1% BSA and the indicated concentrations or concentration ranges of the enzyme substrates For comparison, published KM values for ATP, L-Glu and tRNAGlu for the E coli enzyme are shown [18] ATP (mM) Mt-GluRS Ec-GluRS L-Glu 0.01–2.0 1.0 1.0 Varied 0.5 0.03–2.0 0.5 (mM) tRNAGlu (lM) kcat (min)1) KM 3.6 3.6 0.45–4.0 16.3 ± 0.4 129.0 ± 28.0 24.5 ± 1.5 0.08 2.7 0.7 0.25 0.10 0.16 Varied Varied Alternate substrates and inhibitors of Mt-GluRS Several analogs of the enzyme substrates were tested as alternate substrates or inhibitors of Mt-GluRS (Tables S1 and S2) Mt-GluRS was found to be very specific for the amino acid substrate l-Gln (2–5 mm) 1000 kcat 100 10 0.1 100 kcat/K L-Glu Determination of the apparent kcat and KM for ATP, l-Glu (Kl-Glu) and tRNAGlu was carried out at 37 °C under conditions detailed in Materials and methods and in the legend to Table The quality of data is shown in Fig S3 KM values for ATP and tRNA are of the same order of magnitude as those reported for E coli glutamyl–tRNA synthetase (Ec-GluRS) [18], which we here use as the reference GluRS The Kl-Glu value is very high so that it remains poorly defined and it may be ‡ 2–3 mm, i.e at least $ 20-fold higher than the corresponding value reported for Ec-GluRS The kcat extrapolated at infinite l-Glu concentration at saturating concentrations of the other substrates (129 ± 28 min)1), within the limits imposed by the high value of the KL-Glu that prevents an accurate estimate of this parameter, is now of the same order of magnitude of that reported for Ec-GluRS ($ 100 min)1) [17] The high Kl-Glu value of Mt-GluRS, compared with Ec-GluRS, did not depend on the pH at which the activity assays were carried out Indeed, kcat and Kl-Glu values were determined between pH 6.5 and 8.5 in the presence of fixed concentrations of the other substrates (Fig 1) kcat values were found to increase as a group with an apparent pKa < dissociated to reach a constant value above pH 7.3 The kcat ⁄ Kl-Glu profile instead showed a plateau at pH values between 6.5 and 7.5 and decreased at high pH as a group with a pKa value > deprotonated Because of the high cost of l-[U14C]Glu and the need to maintain the ionic strength of the assay relatively low, enzyme activity was routinely measured in the presence of 0.5 or mm l-Glu ± 0.01 mM ± 0.8 mM ± 0.2 lM mM mM lM 10 pH Fig pH dependence of the steady-state kinetic parameters kcat and kcat ⁄ KL-Glu of Mt-GluRS The apparent kcat (in min)1) and kcat ⁄ KL)1 )1 Glu (in ỈmM ) values of the reaction catalysed by Mt-GluRS (6.3 nM) were determined at 37 °C in 35 mM Hepes ⁄ NaOH buffer at the indicated pH values in the presence of mM ATP, 3.6 lM tRNAGlu, 10 mM MgCl2, 25 mM KCl, mM dithiothreitol, 10% glycerol 0.1% BSA and varying L-[U14C]Glu kcat values were fitted to Eqn (13), assuming that kcat increases to a limiting value of 110 ± 1.0 min)1 at high pH as a single group with pKa of 6.2 ± 0.03 deprotonates kcat ⁄ KL-Glu values fitted well fitted with Eqn (14) assuming that the parameter decreases from a limiting value of 70 ± 5.0 min)1ỈmM)1 as a group with a pKa value of 8.7 ± 0.23 deprotonates and 2-oxoglutarate (1–5 mm) did not inhibit the reaction (Table S1) Furthermore, l-Gln could not efficiently substitute for l-Glu as the substrate Indeed, in the presence of mm l-Gln the apparent turnover number was 0.03 min)1, i.e 0.1% of that measured in the presence of mm l-Glu (Table S1) Mt-GluRS is FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1401 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al Requirement of tRNA for the adenylation of l-Glu in MtGluRS GluRS, together with GlnRS, ArgRS and class I LysRS, are the only aaRS that require bound tRNA Table Inhibition of Mt-GluRS by glutamol-AMP and pyrophosphate Activity assays were carried out at 37 °C in 35 mM Hepes ⁄ NaOH, pH 7.3, in the presence of mM dithiothreitol, 10% glycerol, 0.1% BSA, 10 mM MgCl2, 25 mM KCl, Mt-GluRS (6.3 nM in the 150 lL assay mixture) When the substrate concentrations were held constant they were: mM ATP, 0.5 mM L-Glu and 3.6 lM tRNA The inhibition pattern was established throught the best fit of the data to Eqns (10–12) describing competitive (C), noncompetitive (NC) and uncompetitive (UC) inhibition, respectively Inhibitor Glutamol-AMP Varied substrate ATP L-Glu Pyrophosphate 1402 tRNA ATP L-Glu Pattern C C UC NC NC Kis (lM) Kii (lM) 1.5 ± 0.4 3.9 ± 1.0 31.4 ± 7.6 12.5 ± 3.7 3.9 ± 0.7 27 ± 15.4 101 ± 54 in order to form the aminoacyl-AMP intermediate from ATP and the free amino acid [2,4] To establish the requirement of tRNA for the aminoacyl–adenylation reaction, Mt-GluRS was incubated with either l-[14C]Glu or [3H]ATP under various conditions, and the reaction components were identified and quantified after chromatographic separation on a MonoQ column (Fig S5) Only in the presence of both tRNA and l-Glu was the appearance of [3H]AMP observed That the radioactivity associated with the elution position of AMP did not correspond to Glu-AMP was tested by carrying out the same experiments in the presence of l-[U14C]Glu (not shown) The kinetics of [3H]AMP formation were also determined (Fig 2) The amount of [3H]AMP formed at early incubation times matched well with that of l-[U14C]Glu–tRNAGlu formed in parallel filter-binding assays At later reaction times the amount of AMP formed exceeded that of Glu–tRNA, presumably due to recycling of tRNA derived from [AMP], µM; [Glu-tRNA] µM also highly specific for the nucleotide substrate ATP could not be substituted as the substrate by b,c-methylene-ATP, despite the presence of the hydrolysable a,b-phoshoanhydride bond a,b-Methylene-ATP and b,c-methylene-ATP were not inhibitors of the reaction (Table S2) nor were AMP and its analog decoyinine [19] For comparison a,b-methylene-ATP was found to be an inhibitor of Ec-GluRS, competitive with ATP (Ki $ 0.45 mm) [20] With Ec-GluRS AMP was a noncompetitive inhibitor with respect to ATP and l-Glu with Ki values in the mm range, as deduced by the data presented in Kern and Lapointe [20] On the contrary, the glutamyl-AMP analog glutamol-AMP (GoA) [21] and pyrophosphate (PPi) (but not a series of PPi analogs) were potent inhibitors of Mt-GluRS (Table S2) Several divalent cations were also tested as substitutes for Mg2+ or inhibitors None could replace Mg2+ in the reaction, and they all acted as mild inhibitors (Table S3) The inhibitory effect of PPi and GoA was studied in greater detail PPi was found to be a noncompetitive inhibitor with respect to both ATP and l-Glu with Ki values in the 10–100 lm range (Table and Fig S4) GoA was a competitive inhibitor with respect to both l-Glu and ATP with Ki values of $ and 1.5 lm, respectively These values are similar to those reported for the Ec-GluRS ($ lm with respect to both substrates) [21] GoA was instead uncompetitive with respect to tRNA (Ki $ lm) 0 10 15 Time (min) Fig Kinetics of [3H]AMP formation during Mt-GluRS reaction as determined by chromatographic separation of the reaction components GluRS (0.67 lM) was incubated at 37 °C in 35 mM Hepes ⁄ NaOH buffer, pH 7.3, 10% glycerol, mM dithiothreitol, mM [2,5Â,83H]ATP (33 300 dpmặnmol)1), 10 mM MgCl2, 25 mM KCl, mM L-Glu and 3.6 lM tRNAGlu (s) in a final volume of 150 lL After 1–10 min, cold water was added (2 mL) and a mL sample was rapidly injected onto a MonoQ column equilibrated in 20 mM triethanolamine ⁄ HCl buffer, pH 7.7, and developed by increasing the KCl concentration in the same buffer Fractions (1 mL) were collected and the radioactivity was measured by scintillation counting The concentration of [3H]AMP formed in the assays at any given time was calculated from the amount of radioactivity present in the AMP elution peak (Fig S5) In separate experiments the timecourse of [3H]AMP formation in assays lacking L-Glu (d) or L-Glu and tRNAGlu (h) was also measured The kinetics of Glu–tRNAGlu formation ( ) determined using the filter-binding assay in the presence of 14 L-[U C]Glu and unlabelled ATP under identical conditions is shown for comparison Note that at long incubation times, the amount of AMP formed exceeds that of Glu–tRNA due to recycling of tRNA because of the spontaneous hydrolysis of Glu–tRNA FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al M tuberculosis glutamyl–tRNA synthetase 25 Fig Kinetics of [3H]AMP and [3H]ADP formation from ATP Assays were set up in 35 mM Hepes ⁄ NaOH buffer, pH 7.3, 10% glycerol, mM dithiothreitol, mM [2,5Â,83H]ATP (33 300 dpmặnmol)1), 10 mM MgCl2, 25 mM KCl, mM L-Glu, 0.004% BSA and 3.6 lM tRNAGlu in a final volume of 150 lL and incubated at 37 °C in the presence of different Mt-GluRS concentrations [6.7 nM (circles), 76.3 nM (squares) and 822 nM (triangles)] At different times, 10 lL aliquots were rapidly applied onto poly(ethyleneimine)–cellulose sheets, subjected to TLC and quantification of radiolabelled ADP and AMP The kinetics of [3H]AMP and [3H]ADP formation in the absence of tRNAGlu (black symbols) or of both L-Glu and tRNAGlu (grey symbols) were also determined in parallel samples (A) Time-course of AMP formation in the complete assay mixture at the three different enzyme concentrations (open symbols) In the absence of tRNAGlu no AMP formation above background was detected (closed symbols) Similar results were obtained in the absence of both tRNAGlu and L-Glu (not shown) Note that the reaction velocity is independent of the Mt-GluRS concentration because the formation of AMP is monitored at long incubation times and with high Mt-GluRS concentrations when recharging of tRNA derived from hydrolysis of Glu–tRNAGlu is being observed (B, C) At increasing Mt-GluRS concentrations, formation of ADP from ATP could be detected at rates that were essentially independent from the presence of the enzyme substrates The time-course of ADP formation in the complete assay mixture (not shown) was similar to that obtained in the absence of tRNAGlu (B) A AMP (µM) 20 15 10 –5 25 B ADP (µM) 20 15 10 –5 25 C ADP (µM) 20 15 10 –5 60 120 180 Time (min) spontaneous hydrolysis of Glu–tRNA [22] These results were confirmed by separating the reaction components using TLC (Fig 3A) In these experiments, we also observed ADP formation (Fig 3B,C) at a rate that was dependent on the enzyme concentration, but independent of the presence of l-Glu and tRNA This ATP hydrolysing activity is very low (0.135 min)1 when calculated from the kinetics of ADP formation in the absence of tRNA with 0.8 lm Mt-GluRS) (Fig 3B) and it is unlikely to represent a physiologically relevant side reaction PPi ⁄ ATP exchange reaction of Mt-GluRS Evidence for the presence of the Glu-AMP intermediate in GluRS and in other aaRS has been obtained by studying the [32P]PPi ⁄ ATP exchange reaction [2,4,23,24] Thus, Mt-GluRS was incubated under various conditions with [32P]PPi and the reaction components were separated by TLC [32P] associated with the various compounds was quantified using a phosphoimager (Fig S6) No radioactive species other than PPi and minor amounts of Pi were observed when the enzyme was incubated in solutions lacking one of the enzyme substrates When all three substrates were present, only formation of [32P]ATP was observed In kinetic experiments, the rate of formation of [32P]ATP increased at increasing concentrations of [32P]PPi (Fig 4) As expected from the observed inhibitory effect of PPi on the tRNA charging reaction, the velocity of [32P]ATP formation was inversely proportional to that of l-[U14C]Glu–tRNA production in parallel filter-binding assays (not shown) The kcat of [32P]ATP formation was 1100 ± 174 min)1 This value should be compared with that calculated for the tRNAGlu charging reaction under similar conditions (90 min)1) Is Mt-GluRS a discriminating or a non-discriminating GluRS? According to analyses of the M tuberculosis genome, Mt-GluRS is predicted to be of the nondiscriminating type (see above for details) Attempts to produce M tuberculosis tRNAGlu and tRNAGln (in vivo or FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1403 M tuberculosis glutamyl–tRNA synthetase 800 A v/E (min–1) [32P]A (nmol) ATP 0 Time (min) S Paravisi et al production in both Luria–Bertani (Fig S1) and M9 medium (not shown) B 400 0.0 Structural studies on Mt-GluRS in solution 0.5 [PPi] (mM) 1.0 Fig Kinetic parameters of PPi ⁄ ATP exchange reaction The time-course of the incorporation of [32P]PPi into ATP was determined as a function of PPi concentration (empty circle, 0.01 mM; full circle, 0.1 mM; empty square, 0.5 mM; full square, 1.0 mM) in 100 mM Hepes ⁄ NaOH buffer, pH 7.2, containing 0.3% glycerol, mM ATP, 16 mM MgCl2, mM L-Glu, 25 mM KCl, 0.01–1 mM Na-[32P]PPi (231 113 dpmỈnmol)1), 3.6 lM tRNAGlu and Mt-GluRS (7.4 nM) in a final volume of 50 lL, at 37 °C Aliquots (1 lL) of the reaction mixtures were withdrawn before and at different times after tRNA addition They were rapidly applied onto the poly(ethyleneimine)–cellulose sheets, which were developed immediately After quantitation of [32P]ATP formed at the different times (left), calculation of the initial reaction velocity and correction for the amount of enzyme present, the rates of [32P]ATP formation at the different PPi concentrations were fitted to the Michaelis–Menten equation to calculate the apparent kcat (1101 ± 174 min)1) and KM for PPi (0.4 ± 0.17 mM) of the reaction (right) For comparison, the velocity of Glu–tRNA formation under similar substrate concentrations was calculated to be 89 min)1 in vitro) in quantities sufficient to carry out kinetic assays have not been successful, yet Thus, in order to study the discriminating or nondiscriminating nature of Mt-GluRS we used the toxicity test developed by Baick et al [25] Overproduction of the nondiscriminating Bacillus subtilis GluRS (Bs-GluRS) in E coli cells, which lack the Glu–tRNAGln amidotransferase, was found to be toxic Supplementing the medium with l-Gln protected the cells, presumably by allowing the endogenous GlnRS to saturate the tRNAGln with l-Gln, thus avoiding the misacylation reaction Diluted cultures of E coli BL21(DE3) cells containing pETGTS1 were plated on Luria–Bertani or M9 medium (to measure the cells vitality) or medium containing ampicillin (to count cells containing the plasmid) in the absence or presence of isopropyl thio-b-d-galactoside (IPTG) (to establish the toxicity of the overproduction of Mt-GluRS) The effects of l-Gln (2.5–25 mm, to relieve toxicity) and of l-Glu (2.5– 25 mm, to enhance the hypothesized misacylation reaction of tRNAGln) were also tested In none of the conditions (Table S4) was pETGTS1 toxic, nor was the induction of the gene expression This agrees well with the fact that large amounts of soluble Mt-GluRS were produced in E coli BL21(DE3) cells for protein 1404 Several unsuccessful attempts were made to obtain crystals of the protein for X-ray diffraction studies With the aim of gathering structural information (i.e oligomeric structure, conformational flexibility, effect of ligands) we carried out limited proteolysis and small-angle X-ray scattering (SAXS) measurements on Mt-GluRS, using the Ec-GluRS species as a reference protein and the available high-resolution structures of Tt-GluRS [5,12,13] and Te-GluRS [15] as models Both Mt- and Ec-GluRS were found to be more sensitive to trypsin than to chymotrypsin, with Mt-GluRS being significantly more sensitive than Ec-GluRS to the given protease Incubation of Mt-GluRS with chymotrypsin 0.1% (w ⁄ w) led to the formation of a limited number of protein fragments with five main species (bands M1-M5 of Fig 5), of which M2 (27.5 kDa), M4 (18.4 kDa) and M5 ($ kDa) were stable to further proteolytic attack From the N-terminal sequence and the mass of the fragments, and from the kinetics of the process (Figs S7–S9 and Table S5) we concluded that the main sites of proteolytic cleavage are at the C-terminus of the predicted catalytic domain and in the stem-contact domain [5,12,13,15] By projecting these cleavage sites on the Tt-GluRS structures available, they are found to be close to the ATP-binding site (not shown) Accordingly, GoA (Fig 5) and ATP (not shown) fully protected MtGluRS from chymotryptic degradation Interestingly, MgCl2 was not required for the binding of these nucleotides to GluRS Ec-GluRS was less sensitive than Mt-GluRS to chymotrypsin and a limited number of fragments could be observed using 1% chymotrypsin (Fig 5) Analysis of the proteolytic fragments (Figs S7–S9 and Table S5) indicated that the main proteolytic site in Ec-GluRS is S238 From sequence comparisons (Fig S7), this residue is in the ‘KMSK’ fingerprint of GluRS, which identifies the ATP-binding site [2] At variance with the Mt-GluRS, GoA and ATP had no effect on the proteolytic pattern observed with Ec-GluRS l-Glu did not have an effect on proteolysis with any of the enzymes In order to extract structural information, although at a low resolution, protein samples were analysed by SAXS DLS provided an important set of information preliminary to the SAXS experiments, allowing us to establish working conditions and revealing that Ec- and Mt-GluRS likely differed for their aggregation state (Fig S10 and accompanying text for details) FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al M tuberculosis glutamyl–tRNA synthetase Fig SDS ⁄ PAGE analysis of E coli (upper) and M tuberculosis GluRS (lower) limited proteolysis products The enzymes (1 mgỈmL)1) were incubated with 1% (Ec-GluRS) or 0.1% (Mt-GluRS) (w ⁄ w) Na-tosyl-L-lysyl chloromethyl ketone-treated chymotrypsin in 50 mM Hepes ⁄ NaOH buffer, pH 8.0, in the absence or presence of 0.5 mM GoA at 25 °C in a final volume of 100–200 lL At different incubation times, aliquots (10 lL) were analysed by SDS ⁄ PAGE after chymotrypsin inactivation The mass of the peptides was calculated by comparison with a calibration curve built with the 14–202 kDa molecular mass protein standard mix (*) E1 (45.3 kDa), E2 (30.3 kDa) and E3 (26.6 kDa) are the main proteolysis products obtained with Ec-GluRS (54.0 kDa) From N-terminal sequencing E2 corresponds to the N-terminal fragment of Ec-GluRS and E3 starts at position 238 M1 (28.9 kDa), M2 (27.5 kDa), M3 (23.4 kDa) and M4 (18.4 kDa) derive from Mt-GluRS (54.3 kDa) The N-terminal sequences of M1 and M2 corresponds to the N-terminus of intact Mt-GluRS M4 starts at position 319 Ec-GluRS solutions (0.5–11 mgỈmL)1) yielded scattering patterns consistent with the presence of one species in solution The scattering curves computed from the atomic coordinates of the Tt- or Te-GluRS monomers by program crysol [26] yielded reasonable fits to the experimental patterns (Table S6 and Fig 6A, curve 1) Mt-GluRS yielded more complex SAXS patterns (Fig 6A, curves 2–16) The calculated radius of gyration (Rg) and molecular mass (MM) increased at increasing protein concentration (Table S6), indicating the presence of multiple species in solution At protein concentrations > mgỈmL)1 the calculated Rg stabilized at 3.8 nm suggesting the presence of a dimeric species (Table S6 and Fig 6A, curves and 3) Te-GluRS is a dimer in the crystal form [15], as opposed to Tt-GluRS [5,12–14] However, a poor fit to the data was obtained by assuming for Mt-GluRS a structure similar to that of the Te-GluRS dimer, even taking into account a monomer–dimer equilibrium Therefore, a model Mt-GluRS dimer was built on the basis of the structure of the Tt-GluRS subunit extracted from that of the Tt-GluRS ⁄ tRNA complex (PDB code 1g59) The scattering curve of the symmetric homodimer shown in Fig 6B clearly yielded the best fits to the SAXS data of Mt-GluRS at concentrations > mgỈmL)1 Based on this dimeric model of Mt-GluRS, and using the program oligomer [27], it was possible to quantify the relative distribution of Mt-GluRS monomers and dimers at various protein concentrations, confirming the concentration dependence of the monomer–dimer equilibrium of Mt-GluRS solutions (Fig 6A, curves 2–6 and Table S6) The presence of l-Glu or GoA had no effect on the scattering curves of Mt-GluRS solutions (not shown), whereas ATP, either alone or in the presence of l-Glu, shifted the equilibrium towards the monomer, without causing full conversion (Fig 6A, curves 7–10 and Table S6) By contrast, MgCl2 appears to stabilize the dimer (Fig 6A, FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1405 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al B A Fig SAXS analysis of GluRS oligomerization in solution (A) Scattering profiles of (1) Ec-GluRS (the pattern merged from different concentrations, no oligomerization effect observed); (2–6) Mt-GluRS with no MgCl2 and no ATP for concentrations (mg.mL)1) c = 6.25, 4.16, 3.7, 1.8 and 0.9 (from top to bottom); (7–10) Mt-GluRS with ATP (1 mM) and no MgCl2 at c = 7.85, 4.16, 1.63 and 0.86; (11–14) Mt-GluRS with MgCl2 (0.2 mM) and no ATP, c = 4.4, 3.96, 3.75 and 3.56; (15,16) Mt-GluRS with MgCl2 (0.2 mM) and ATP (1 mM) (c = 3.9 and 3.67) Experimental data are denoted by black dots and the fits from OLIGOMER [27] (or CRYSOL [26] for Ec-GluRS) are shown as red solid lines The curves are appropriately displaced in logarithmic scale for better visualization (B) The dimer composed by two adjacent monomers (shown in red and blue), which yields the best fit to Mt-GluRS data at concentrations > mgỈmL)1 in the absence of ligands The monomer structure was extracted from the crystal structure of Tt-GluRS in complex with tRNA (PDB ID 1g59) curves 11–14 and Table S6) and reduces dissociation into monomers when ATP is added (Fig 6A, curves 15–16 and Table S6) Interaction between M tuberculosis GluRS and GluTR In those bacteria and plants that use the C5 pathway for ALA biosynthesis it has been proposed that GluRS and GluTR may form a complex in order to commit Glu–tRNAGlu to tetrapyrrole biosynthesis Complex formation between GluRS and GluTR has been shown with purified Chlamydomonas reinhardtii enzymes [11] We tested complex formation between the M tuberculosis enzymes by using affinity chromatography 1406 Although homogeneous preparations of Mt-GluRS can be obtained as described above, all attempts to produce large amounts of the putative Mt-GluTR (Rv0509) in a soluble form in E coli or M smegmatis cells were unsuccessful (not shown) However, cloning of Rv0509 in pET11a or in pET23b (to generate a C-terminally His6-tagged version of Mt-GluTR, Mt-GluTR–His) led to the production of a small amount of soluble protein, which could be increased by co-producing the E coli chaperon proteins DnaJ, DnaK and GrpE from plasmid p20 [28] The identity of Rv0509 with the Mt-GluRS was established indirectly E coli cells overexpressing Rv0509 were red due to the accumulation of heme, which was in part released in the culture medium, as established from the FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al M tuberculosis glutamyl–tRNA synthetase Fig GluRS–GluTR interaction (Upper) The crude extract obtained from the homogenization of E coli BL21 (DE3, pGTR, p20) cells (2 g) that contained the native Mt-GluTR, was incubated with Mt–His6GluRS (2 mg) for 30 at °C and 10 r.p.m on a rotary shaker Two millitres of a 50% Ni-nitrilotriacetic acid-Sepharose suspension in 20 mM Hepes ⁄ NaOH buffer, pH 8.0, 10% glycerol, mM b-ME was added After h the suspension was poured into a chromatographic column and the packed resin was extensively washed with the equilibration buffer The column was developed with a 0–100 mM imidazole gradient in 10 mM steps followed by a final wash with 500 mM imidazole Aliquots of the collected fractions were denatured for SDS ⁄ PAGE The gels were stained with Coomassie Brilliant Blue and destained (Middle) The cell extract was substituted by a crude extract of E coli BL21 (DE3, pET23b, p20) cells (Lower) The His6–GluRS solution was substituted by the same volume of buffer In all gels the fractions eluted with 50–100 mM imidazole showed no detectable proteins so that the corresponding lanes are not shown The column flow-through has also been omitted The dots mark an E coli protein that migrates just below Mt-GluTR In the upper gel, the white box highlights the fraction containing both GluRS and GluTR, whose spectrum and DLS signal are shown in Fig S11 The migration positions of GluTR and GluRS, were determined by comparison with those of a homogeneous sample of GluRS (S) and of a sample enriched in GluTR (R) obtained by solubilizing inclusion bodies from overproducing cells The band corresponding to GluTR has also been identified by western blots and immunodecoration with anti-GluTR IgG The star indicates the standard proteins, with the corresponding mass shown on the side of the gels (in kDa) absorbance spectra of crude extracts and culture medium Such a red phenotype is expected for the overproduction of the enzyme catalysing the first and rate-limiting step of tetrapyrrole biosynthesis [8] Interestingly, the red phenotype was lost when the C50S and C50A variants of Rv0509 were overproduced (not shown) C50 of Rv0509 corresponds to the catalytically essential C48 of Methanopyrus kandlerii GluTR, which together with the E coli form is the best characterized GluTR [29–32] Attempts to purify Mt-GluTR led to the isolation of aggregates of > MDa Therefore, the interaction between M tuberculosis GluRS and GluTR had to be studied using purified Mt-GluRS and Mt-GluTR forms contained in crude extracts of overproducing cells Addition of His6–GluRS to a crude exctract of cells that had produced Mt-GluTR followed by affinity chromatography on Ni-nitriloacetate–Sepharose allowed us to demonstrate formation of the GluRS-GluTR complex (Fig 7) Similar results were obtained by adding homogenous GluRS to a crude extract of cells that had produced Mt-GluTR–His (not shown) Interestingly, all column fractions containing Mt-GluTR (Fig 7) or its His6-tagged variant (not shown), exhibited an absorbance spectrum consistent with the presence of a protein-bound heme cofactor (Fig S11A) Furthermore, the DLS signal of the fractions containing Mt-GluTR (Fig 7, top, boxed lane) showed a single, although broad, peak (r = 5.2 nm) corresponding to a mass of only 180 kDa (Fig S11B) This finding suggests that complex formation with GluRS and GluTR may isolate and stabilize a soluble GluTR form FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1407 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al Discussion Rv2992c gene product was demonstrated to encode Mt-GluRS, which is capable of charging E coli tRNAGlu with l-Glu The enzyme can be obtained in large quantities and in a soluble and stable form using a four-step purification procedure based on previously described methods [17,18,33] Mt-GluRS exhibits properties similar, but not identical, to those of the well-characterized GluRS from E coli, which we used as the prototype of bacterial GluRS The turnover number is similar to that reported for Ec-GluRS, as are the KM values for ATP and tRNAGlu However, KL-Glu was found to be $ 20fold higher for Mt-GluRS than for the E coli enzyme Such a difference cannot be ascribed to a different pH dependence of the reaction, but rather to a difference between the enzymes Like other GluRS, the M tuberculosis enzyme requires bound tRNAGlu to carry out the formation of the Glu-AMP intermediate indicating that for Mt-GluRS also binding of tRNA might induce the conformational change observed with Tt-GluRS that switches the binding mode of ATP to a productive one [5] Mt-GluRS is similar to other GluRS in that it catalyses the PPi ⁄ ATP exchange reaction, indicating that reaction steps linking the enzyme ⁄ tRNA ⁄ Glu ⁄ ATP complex to yield the enzyme ⁄ tRNA ⁄ GluAMP complex are reversible The kcat value measured during the PPi ⁄ ATP exchange reaction is $ 10-fold higher than that measured for the tRNA aminoacylation reaction, indicating that transfer of l-Glu from Glu-AMP to the tRNA is slower than pyrophosphorolysis of the intermediate to yield ATP and l-Glu A similar conclusion was reached for Ec-GluRS However, for Ec-GluRS, it has been reported that the pH dependence of the PPi ⁄ ATP exchange reaction follows an inverse profile with respect to that of the tRNA charging activity, so that the ratio between the velocities of the exchange and charging reactions was $ 30 at pH 6.2, but only 1.5 at pH 7.4 and 0.3 at pH 8.6 [22,24] Thus, our finding that the PPi ⁄ ATP exchange is $ 10-fold faster than tRNA charging at pH 7.2, while supporting a similar reaction mechanism for the two enzymes, suggests a different pH dependence for the individual reactions steps, perhaps reflecting differences in the fine structure of their active sites Mt-GluRS was found to be very specific for the amino acid and the nucleotide substrate It could use l-Gln instead of l-Glu to charge tRNAGlu only at a very low rate Neither l-Gln nor 2-oxoglutarate are inhibitors Despite the presence of the a-b hydrolysable bond, b,c-methylene-ATP could not replace ATP as the substrate Furthermore, the a,b- and b,c-methylene analogs of ATP tested, and AMP and its analog decoinine did not inhibit Mt-GluRS This is at variance with Ec-GluRS which was inhibited by both a,b-methylene-ATP and AMP, although with Ki values in the mm range [20] As for the E coli enzyme, PPi, which binds to the enzyme ⁄ tRNA ⁄ Glu-AMP intermediate, was found to be a noncompetitive inhibitor with respect to both l-Glu and ATP GoA was competitive with respect to both Glu and ATP, but uncompetitive with respect to tRNAGlu, with Ki values of the same order of magnitude as those reported for Ec-GluRS [21] GoA is one of the glutamyl-AMP analogs being developed as a GluRS inhibitor, and it will be of interest to test them on Mt-GluRS in future studies of potential novel antitubercular drugs, which are the long-term aim of this project [21,34,35] The precise definition of the kinetic mechanism of Mt-GluRS was outside the scope of this work, particularly in light of the complexity of the reaction, as shown by the elegant work done with the E coli enzyme [20,22–24] and the expected overall similarity between Mt- and Ec-GluRS However, our data are consistent with a minimal reaction scheme in which tRNA binding to the free enzyme is followed by an activatory conformational change [5] and random binding of l-Glu and ATP to the latter species (Scheme 1) In particular, the competitive inhibition pattern observed with GoA versus l-Glu and ATP is diagnostic for the random sequential portion of the Scheme Minimal kinetic scheme of the Mt-GluRS reaction 1408 FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al kinetic mechanism as discussed recently [36] for bisubstrate inhibitors In the absence of tRNA and l-Glu, Mt-GluRS was found to hydrolyse ATP to ADP + Pi, although the reaction velocity was only 0.1% that of the physiological tRNAGlu charging reaction, under the same conditions, leading to the conclusion that this reaction is not biologically relevant Mt-GluRS differs from Ec-, Tt- and Te-GluRS for the oligomeric state in that its monomer exists in solution in equilibrium with the dimeric species At the low concentrations used in the activity assays, the enzyme monomer should prevail, indicating that this species is catalytically active By contrast, Ec-GluRS and Tt-GluRS [5,12–14] appear to be strictly monomeric, whereas the crystal structure of the Te-GluRS shows dimers [15] By using SAXS and the Tt- and Te-GluRS structures for rigid body modeling, it was determined that the overall shape of the Mt- and Ec-GluRS subunits are similar to each other and to those of Tt- and Te-GluRS SAXS sensitivity is not sufficient to distinguish among the conformations of Tt-GluRS bound to the different ligands and that of the Te-GluRS subunit The crystallographically detected conformations are indeed catalytically significant, but structurally minor, implying rotations of domains of just a few degrees (e.g 7° interdomain rotations upon tRNA binding to the Tt-GluRS and local limited rearrangements in the active site) [5] Interestingly, the Mt-GluRS dimer found in solution appears to differ from that found in the Te-GluRS crystals [15] However, by fitting the SAXS curves a model could be built, which indicates that this species may be catalytically active because the tRNA binding surface and ATP and l-Glu binding sites are solvent accessible Despite the structural similarity of the enzyme subunits, Mt-GluRS is significantly more sensitive than Ec-GluRS to proteolysis, suggesting greater conformational flexibility In both enzymes, the sites sensitive to chymotryptic attack are next to the ATP-binding site However, only in the case of Mt-GluRS are the chymotrypsin-sensitive sites protected by ATP and GoA, highlighting another difference between the enzymes Interestingly, ATP and GoA had a similar effect on the proteolytic pattern of Mt-GluRS, but only ATP appeared to stabilize the monomeric form, as established by SAXS The flexibility of Mt-GluRS coupled to the monomer ⁄ dimer equilibrium may be the reason for the failure to obtain crystals suitable for determination of the Mt-GluRS structure by X-ray diffraction Despite M tuberculosis genome analysis indicating that Mt-GluRS is of the discriminatory type (see above for details), overproduction of Mt-GluRS in E coli cells, M tuberculosis glutamyl–tRNA synthetase which lack the Glu–tRNAGln amidotransferase needed to correct the misacylation of tRNAGln caused by a NDGluRS, is not toxic These results lead to the conclusion that Mt-GluRS ⁄ tRNAGln recognition is species specific Sequence analyses may provide a rationale for the discriminating behaviour of Mt-GluRS in E coli Studies on Tt-GluRS indicated that discriminating and nondiscriminating GluRS can be distinguished on the basis of the presence of a specific Arg residue (Arg358 in Tt-GluRS, Arg350 in Ec-GluRS) in the anticodon recognition region of GluRS [12] (Fig S7) Indeed, its substitution with a Gln, the residue found at the equivalent position in Bs-GluRS, conferred nondiscriminating properties on Tt-GluRS [12] Furthermore, Te-GluRS, a ND-GluRS, contains Gly366 in the position equivalent to Arg358 of Tt-GluRS [15] However, site-directed mutagenesis of the two GluRS isoforms of Helicobacter pylori and comparative sequence analyses [37] indicated that the Arg residue is not sufficient to distinguish between D- and NDGluRS but, more likely, several residues are important One was identified as a Thr (Thr444 in Tt-GluRS) found in most D-GluRS, which is often substituted by Gly, Ala, Ser (e.g Gly454 in Te-GluRS) in NDGluRS A third candidate was found by Schultze et al [15] who observed that in Tt-GluRS Arg358 forms a salt bridge with Glu443, which is not found in several ND-GluRS even when they have an Arg residue equivalent to Tt-GluRS Arg358 Comparison of the sequence of Mt-GluRS with those of the T thermophilus, T elongatus, E coli and B subtilis enzymes showed that Arg372 of Mt-GluRS is at a position equivalent to that of Arg358 in Tt-GluRS, and Ser461 substitutes Thr444 (Fig S7) In Mt-GluRS the preceeding residue is Val460, which corresponds to Glu443 of Tt-GluRS and His453 of Te-GluRS Thus, Mt-GluRS seems to obey to the rules established previously [15,37] for a ND-GluRS However, these rules not seem sufficient to predict the discriminating properties of a GluRS In particular, the discriminating Ec-GluRS has a Gln437–Ser438 pair where a Glu–Thr is expected That Mt-GluRS may be of the nondiscriminating type in M tuberculosis (as supported by genome analyses), but its sequence shares features with the discriminating Ec-GluRS (Arg372, Val460 and Ser461 in Mt-GluRS versus Arg350, Gln437 and Ser438 in Ec-GluRS), might explain the absence of toxicity of its overproduction in E coli where it behaves as a D-GluRS like the endogenous enzyme Finally, we found that M tuberculosis GluRS and GluTR can form a complex confirming the results obtained with C reinhardtii enzymes [11] and FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1409 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al supporting the concept that in M tuberculosis formation of this complex may regulate the flux of Glu– tRNAGlu toward tetrapyrrole biosynthesis as opposed to that of proteins During the course of these studies we also demonstrated that Mt-GluTR contains bound heme, a property previously ascribed only to the planttype enzyme [38,39] Finally, the fact that Mt-GluTR isolated using Mt-GluRS as bait does not seem to aggregate or precipitate also opens the way to the isolation and subsequent characterization of this enzyme responsible for heme biosynthesis, which has also been demonstrated to be essential in M tuberculosis [16] the same restriction enzyme and purified, yielding pETGTS1 The NdeI fragment was also cloned into pET28b (Novagen) digested with the same enzyme The resulting plasmid (pETGTS2) encoded a fusion between an N-terminal His6 tag and the Rv2992c coding region with a ten residues spacer between the sixth His residue and the start codon of the predicted Rv2992c gene product The resulting protein is indicated as His6-GluRS The insert of all plasmids and the adjacent regions were sequenced by PRIMM srl (Milan, Italy) Materials and methods pETGTS1 and pETGTS2 were used to produce the Rv2992c gene product or the N-terminally His6-tagged variant, respectively, in E coli BL21(DE3) cells grown at 25 °C in Luria-Bertani medium containing 0.1 mgỈmL)1 ampicillin Overexpression of the heterologous gene was induced at an D600 value of 0.7 by adding IPTG to a final concentration of 0.1 mm After 19 h, cells were harvested by centrifugation at 6000 g and °C for 15 The cell pellet was washed with 0.9% NaCl and stored at )20 °C until protein purification Chemicals and materials Restriction endonucleases were obtained from GE Healthcare (Chalfont St Giles, UK) and Promega (Madison, WI, USA) Unless otherwise stated, chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) or Merck (Whitehouse Station, NJ, USA) TLC poly(ethyleneimine)– cellulose sheets with fluorescent indicator (254 nm) were from Macherey-Nagel (Duren, Germany) ¨ Production of E coli GluRS in E coli BL21(DE3) cells Cloning of M tuberculosis GluRS gene M tuberculosis Rv2992c, corresponding to the putative gltX gene encoding GluRS, was amplified by PCR using cosmid BAC Rv30 from the Institut Pasteur collection as the template in the presence of synthetic oligonucleotides pairs Primer 1: 5¢-AAGAAGAAGCATATGTCACCGTGCCCG ACCAGCTG-3¢ Primer 2: 5¢-AAGAAGAAGCATATGACCGCCACGG AAACAGTCCGG-3¢ The primers introduced NdeI sites (underlined) for cloning of the amplified fragment into pET11a (Novagen, San Diego, CA, USA) digested with NdeI The GTG start codon was also changed into an ATG (bold in Primer 1) by the insertion of the NdeI restriction site PCR was set up by mixing BAC Rv30 (30 ng), dNTPs (50 lm each), primer and (24 pmol each) and PfuTurbo Taq polymerase (Stratagene, La Jolla, CA, USA) (15 U) in 20 mm Tris ⁄ HCl buffer, pH 8.8, 10 mm KCl, 10 mm (NH4)2SO4, mm MgSO4, 0.1% Triton X-100 and 0.1 mgỈmL)1 BSA PCR conditions were as follows: cycle 1, at 95 °C; cycles 2–36, at 95 °C, 30 s at 60 °C and at 72 °C; cycle 37, at 72 °C The amplified 1500 bp fragment was purified using the QiaQuik Gel Extraction Kit (Qiagen, Venlo, NL, USA) according to the manufacturer’s instructions, precipitated and digested with NdeI After purification by agarose gel electrophoresis the fragment was ligated with pET11a that had been digested with 1410 Production of M tuberculosis GluRS in E coli BL21(DE3) cells Plasmid pET-ERS was a kind gift of J Lapointe (Univer´ ´ site Laval, Quebec, Canada) It was transformed into E coli BL21(DE3, pLysS) cells, which were grown in Luria–Bertani medium containing 60 lgỈmL)1 kanamycin and 34 lgỈmL)1 chloramphenicol at 37 °C until D600 reached 0.3 Overproduction of the Ec-GluRS was induced by adding IPTG at a final concentration of 0.1 mm After h, cells were harvested and stored as described above Determination of protein concentration Protein concentration of crude extracts was determined by the biuret method [40] and that of purified samples using the Bradford Reagent (Amresco, Solon, OH, USA) [41] BSA (Sigma) was used as the standard Using an electrophoretically homogeneous protein preparation it was determined that a mgỈmL)1 Mt-GluRS solution absorbs 0.79 ± 0.064 at 280 nm (average of 10 determinations), a value similar to that reported for the Ec-GluRS (e280 = 0.87) [18] To calculate the enzyme concentration a mass of 53 685 was used for Mt-GluRS by taking into account the post-translational removal of Met-1 to yield the predicted 489 residues protein A mass of 55 876 was used for His6–GluRS (509 residues after removal of Met-1) A mass of 53 669 was used for Ec-GluRS (470 residues for the mature protein) FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al Purification of Mt-GluRS The procedures of Lin et al [17], Lapointe et al [18] and Kern et al [18,33] for Ec-GluRS were combined to obtain homogeneous preparations of Mt-GluRS Purification consisted of: (a) resuspension of 10–20 g cells in 20–40 mL 10 mm (K)PO4 buffer, pH 7.5, 10% glycerol, mm phenylmethanesulfonyl fluoride, mm dithiothreitol, and cell disruption by sonication and centrifugation at 25 500 g for h at °C; (b) poly(ethylene glycol) 6000 (7%, w ⁄ v) ⁄ dextran (1.4%, w ⁄ v) partitioning of the crude extract; (c) recovery of the top poly(ethylene glycol)-rich phase after centrifugation at 17 500 g for 20 min; (d) chromatography on a first Q-Sepharose ion-exchange column (1.5 · 11.3 cm, 20 mL; GE Healthcare) equilibrated in buffer A (20 mm Tris ⁄ HCl, pH 7.4 25 °C, 10% glycerol, mm dithiothreitol), eluted with buffer A + 0.2 m NaCl (5 vol) and a 0.2– 1.0 m NaCl gradient in buffer A (20 vol, mLỈmin)1); (e) concentration of the pooled GluRS-containing fractions by ultrafiltration in an Amicon apparatus (Millipore, Billerica, MA, USA) equipped with a YM10 membrane, and dialysis against buffer B (50 mm Hepes ⁄ NaOH, pH 8.0, 10% glycerol, mm dithiothreitol, L); (f) chromatography on a second Q-Sepharose column (1.5 · 8.49 cm, 15 mL) equilibrated in buffer C (20 mm (K)PO4 buffer, pH 7.5, 10% glycerol, mm dithiothreitol), and eluted with buffer C (1 vol) followed by a gradient in which the (K)PO4 concentration was varied from 20 to 250 mm and the pH from 7.5 to 6.5 in 20 vol at a flow-rate of mLỈmin)1; (g) concentration of the GluRS-containing fractions by ultrafiltration to $ 20 mgỈmL)1 and mL; and and (h) dialysis against L of buffer B (5 h) followed by dialysis against 0.5 L of buffer B containing 50% glycerol (14 h) The enzyme (typically 40 mgỈmL)1) was stored at )20 °C without significant activity loss for up to years GluRS-containing fractions were pooled after each step on the basis of their electrophoretic pattern Mt-GluRS eluted from the first column between 0.2 and 0.3 m NaCl, and from the second at $ 150 mm (K)PO4 and pH 7.2 The same purification procedure was used to obtain Ec-GluRS preparations E coli BL21 (DE3, pETGTS2) cells (12 g) that had overproduced His6–GluRS, were resuspended in 10 mm Hepes ⁄ NaOH, pH 8.0, 10% glycerol, mm b-mercaptoethanol (b-ME), mm phenylmethanesulfonyl fluoride (24 mL), disrupted by sonication and centrifuged Twenty milliliters of a 50% Ni-nitrilotriacetic acid–Sepharose (NovagenMerck, Darmstadt, Germany) suspension equilibrated in the homogenization buffer were added to the crude extract and the suspension was incubated for h at 12 r.p.m and °C on a rotary shaker The resin was packed into a chromatographic column (inner diameter, 1.5 cm), washed with one column volume of the equilibration buffer, vol of the same buffer containing 0.5 m NaCl, vol of buffer containing 0.5 m NaCl and 10 mm imidazole and then developed with a M tuberculosis glutamyl–tRNA synthetase 10–100 mm imidazole gradient in buffer + 0.5 m NaCl (15 vol, mLỈmin)1) The enzyme eluted from this column at $ 70 mm imidazole The His6–GluRS-containing fractions were pooled on the basis of their electrophoretic pattern, concentrated by ultrafiltration and dialysed against buffer B (2 L, for h) and buffer B + 50% glycerol (0.5 L, 19 h) as described for the native enzyme preparation Also in this case, the enzyme (20–40 mgỈmL)1) was stable for years when stored at )20 °C Electrophoretic techniques and western blots SDS ⁄ PAGE was performed according to Laemmli [42] using 12% minigels and a GE Healthcare SE280 apparatus Protein samples were denatured by incubation at 100 °C for 10 in SDS sample buffer [62.5 mm Tris ⁄ HCl, pH 6.8, 2% (w ⁄ v) SDS, 0.001% (w ⁄ v) bromophenol blue, 10% (w ⁄ v) glycerol, 0.8 mm b-ME] added from two- or fourfold concentrated stock solutions (2· SDS sample buffer or 4· SDS sample buffer) After the run, the gels were stained by immersion in 0.1% Coomassie Brilliant Blue in 40% methanol, 10% acetic acid and destained by diffusion in 40% methanol and 10% acetic acid N-terminal sequence, mass and aggregation state of GluRS A Mt-GluRS aliquot was gel filtered through a Sephadex G25 (medium, GE Healthcare) column equilibrated in 10 mm Hepes ⁄ KOH, pH 7.5, and concentrated to 5–10 mgỈmL)1 using a Centricon-10 (Millipore) microconcentrator The protein mass was determined on diluted samples by MALDI-TOF with a Bruker Daltonics Reflex IV instrument (Brucker Daltonics, Bremen, Germany) equipped with a nitrogen laser N-terminal sequencing of GluRS and proteolytic fragments was carried out with an Applied Biosystems (Foster City, CA, USA) Procise Model 491 sequencer using aliquots of the Mt-GluRS solution or protein samples resolved by SDS ⁄ PAGE and electrotransferred onto a Immobilon-PSQ (Millipore) membrane, stained with Coomassie Brilliant Blue and thoroughly destained [43] DLS measurements were carried out using a DynaPro instrument (Protein Solutions, Charlottesville, VA, USA) in a 50 lL quartz cuvette at 17 °C (average of 20 30 s acquisitions, sensitivity 70–100% depending on protein concentration) Data were analysed using the dynapro software (version V5 or V6) The software uses Eqn (7) [44] to calculate the mass of globular protein of 24–110 kDa MW ẳ 4=3ịpNA Rh=FricRatioị3 =SpecVol 7ị where MW is the molecular mass, NA is Avogadro’s number (6.022 · 1023 mol)1), Rh is the radius in cm, SpecVol is the specific volume (0.726 cm3Ỉg)1) and FricRatio is the FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1411 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al frictional ratio (1.25707) Protein samples were centrifuged in a microfuge at 15 000 g for 10 at °C before each measurement Activity assays tRNA charging reaction The tRNA aminoacylation activity of Mt-GluRS was determined at 37 °C by measuring the rate of formation of acidprecipitable l-[U14C]Glu–tRNA as described previously [18,33] Standard assays contained 35 mm Hepes ⁄ NaOH, pH 7.3, 25 mm KCl, mm dithiothreitol (buffer C), 0.5– mm l-[U14C]Glu (11 431 dpmỈnmol)1; GE Healthcare), 10 mm MgCl2, mm ATP, 3.6 lm E coli tRNAGlu, 0.1% BSA and enzyme (typically, 10–100 ng, 0.19–1.9 pmol, 1.24–12.4 nm) in a volume of 150 lL E coli tRNAGlu (55– 60% specific tRNAGlu; Sigma) was resuspended in buffer C to yield a 160 lm stock solution, which was stored in small aliquots at )20 °C The amount of tRNAGlu present in each batch and its stability were checked by quantifying the amount of l-[U14C]Glu–tRNA obtained in assays in which the charging of the tRNA present was brought to completion Mt-GluRS stock solutions (typically 20–40 mgỈmL)1) were first diluted to mgỈmL)1 in 40 mm Hepes ⁄ NaOH, pH 8.0, 10% glycerol The protein concentration was determined with the Bradford reagent at this stage The enzyme solution was then serially diluted to up to 10 lgỈmL)1 in the same buffer containing 0.1% BSA For each assay, the reaction mixture (145.5 lL) lacking tRNA was equilibrated at 37 °C for A 20 lL aliquot was withdrawn and spotted on a · cm square of Whatman 3MM filter paper (GE Healthcare), which was immediately transferred to a beaker containing 10% trichloroacetic acid and kept under vigorous stirring until the end of the assay The reaction was started by adding tRNAGlu (4.5 lL) At different times 20-lL aliquots were withdrawn, spotted on the Whatman 3MM filters, which were transferred into 10% trichloroacetic acid with magnetic stirring At the end of the assay, all filters were transferred to fresh 10% trichloroacetic acid (500 mL, 10 min) Washings in 5% trichloroacetic acid and 95% ethanol, with interval stirring (10 each) followed Dried filters were placed in an mL plastic vial Radioactivity was determined by scintillation counting in a TriCarb 2100-TR (Perkin–Elmer, Wellesley, MA, USA) after addition of mL of Ultima Gold (Perkin–Elmer) scintillation fluid dpm were calculated from cpm using a calibration curve made with a [14C] standard (Perkin–Elmer) The amount of l-[U14C]Glu–tRNAGlu (in nmol) formed in the 20 lL aliquot at the different times was calculated The initial velocity (v) of reactions was determined by interpolating the initial linear portion of the curve of Glu–tRNAGlu formed as a function of time Activity was expressed as apparent turnover number (v ⁄ E in min)1) by taking into account the amount of enzyme (E) present in the 20 lL aliquot (in nmol) 1412 Steady-state kinetic analyses and inhibition studies The apparent kcat and KM values of Mt-GluRS for tRNAGlu, ATP and l-Glu were determined in the tRNA aminoacylation reaction at 37 °C as described above, except that the concentration of one of the substrates was varied and the levels of the others fixed In these assays the amount of enzyme was chosen in order to observe linearity up to 10 min, and aliquots were typically withdrawn at 1, 2, 3, and 10 The grafit 4.3 software package (Erithacus Software Ltd, East Grinstead, UK) was used to fit the v ⁄ E values as a function of the varied substrate concentration (S) to the Michaelis–Menten equation (Eqn 8) after inspection of the Lineweaver–Burk (double reciprocal) plot (Eqn 9), and to obtain the values and the associated errors of the steady-state kinetic parameters [45] v/E = (kcat S)/(KM + S) 8ị v/E = (1/ kcat ịỵKM =kcat )(1/S) 9ị Inhibition studies were performed by measuring the initial velocity of reactions that contained fixed levels of the inhibitor (I), varying concentration of one of the substrates and constant concentration of others After inspection of the Dixon or the double-reciprocal plots, the data were fitted to the equation describing competitive (Eqn 10), noncompetitive (Eqn 11) or uncompetitive (Eqn 12) inhibition In Eqn (10), Kis and Kii are the inhibition constants affecting the slopes and the intercepts of the double reciprocal plots, respectively [45] v/E = (kcat S)/[S + KM (1 + I/Ki ị v/E = (kcat S)/[S(1 + I/Kii ịỵKM (1 + I/Kis ị v/E = (kcat S)/[S(1 + I/Kii ịỵKM Š ð10Þ ð11Þ ð12Þ The pH dependence of the apparent kcat and kcat ⁄ Kl-Glu values was measured at fixed levels of ATP (1 mm) and tRNAGlu (3.6 lm) and varying l-Glu (0.2–2.0 mm) in 35 mm Hepes ⁄ NaOH buffer at pH 6.5–8.5 All other conditions were as stated for the standard activity assay The calculated values of kcat and kcat ⁄ Kl-Glu were fitted to Eqns (13,14), respectively [45] Limit 10pHpKa ị ỵ1 10pHpKa ị 13ị Limit 10pKa pHị ỵ1 10pKa pHị 14ị Yẳ Yẳ In Eqns (13,14), Limit is the pH independent value of the steady-state kinetic parameter under analysis (Y) FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al Chromatographic separation of reaction components using [2,5¢,83H]ATP and L-[U14C]Glu GluRS (0.5–61 lg; final concentration, 0.06–7.5 lm) from a stock solution prepared as described for the tRNA charging reaction was incubated in 35 mm Hepes ⁄ NaOH buffer pH 7.3, mm dithiothreitol, 10% glycerol, mm [2,5Â,83H]ATP (33 300 dpmặnmol)1; PerkinElmer), 10 mm MgCl2, 25 mm KCl, mm l-Glu in the presence or absence of 3.0 lm tRNAGlu (final volume: 150 lL) After 1–20 at 37 °C, aliquots were injected directly or after dilution with cold water onto a MonoQ column connected to an AKTA apparatus (GE Healthcare) equipped with an absorbance detector set at 254 nm The column was equilibrated with 20 mm triethanolamine ⁄ HCl buffer, pH 7.7 [46] Elution was performed by washing the column with 14 vol of the equilibrating buffer and then increasing KCl concentration in the buffer from to 0.3 m in 30 vol and from 0.3 to m in vol After vol of buffer containing m KCl, the column was re-equilibrated in the starting buffer The flow rate was mLỈmin)1 and fractions (1 mL) were directly collected in mL plastic vials The radioactivity was determined by scintillation counting Assays were also carried out under the same conditions using unlabeled ATP and l-[U14C]Glu In preliminary experiments, 200 lL aliquots of ATP, ADP and AMP (1 mm each), l-[U14C]Glu (2 mm) and tRNAGlu (3.0 lm) were loaded onto the MonoQ column to identify their elution positions Control experiments also showed that addition of unlabeled nucleotides, as carrier, to the samples under analyses did not alter the chromatography nor the distribution and recovery of radioactivity TLC separation of reaction components AMP, ADP, ATP, Pi and PPi were resolved by TLC on poly(ethyleneimine)–cellulose sheets as described previously [47] The enzyme (0.05–6.4 lg, 0.9–118 pmol, final concentration, 6–790 nm) was incubated in 35 mm Hepes ⁄ NaOH buffer, pH 7.3, 10% glycerol, mm dithiothreitol, 25 mm KCl, mm [2, 5Â,83H]ATP (33 300 dpmặnmol)1; PerkinElmer), 10 mm MgCl2, mm l-Glu, 0.004% BSA in the presence or absence of 3.6 lm tRNAGlu (final volume: 150 lL) Parallel samples, lacking one or more of the components were also set up At different times of incubation at 37 °C 10 lL aliquots were removed and applied onto the poly(ethyleneimine)-cellulose sheets, which were immediately developed with 0.75 m NaH2PO4 ⁄ H3PO4 buffer, pH 3.4, as the mobile phase For optimal resolution and minimal background, the poly(ethyleneimine)–cellulose sheets had to be pre-developed in 1.75 m K2PO4 ⁄ H3PO4 buffer, pH 3.4, washed with water, dried and stored at °C for at least 19 h [48] Spots corresponding to AMP, ADP and ATP were identified with a UV lamp at 254 nm Strips corresponding to the sample lanes were cut into 1.5 cm M tuberculosis glutamyl–tRNA synthetase squares and transferred to mL vials for scintillation counting [32P]PPi ⁄ ATP exchange assay Incorporation of [32P]PPi into ATP was determined in reaction mixtures containing 100 mm Hepes ⁄ NaOH, pH 7.2, mm ATP, 16 mm MgCl2, mm l-glutamate, 0.3% glycerol, 25 mm KCl, mm Na-[32P]-PPi (from a 85 mm stock solution, 231 113 dpmỈnmol)1), 3.2 lm tRNAGlu and Mt-GluRS (0-0.7 lm) [23] After incubation at 37 °C for different times, 1–10 lL aliquots were applied onto the poly(ethyleneimine)–cellulose sheets, which were developed as described above The migration position of the nucleotides was determined by irradiation with UV light It was marked on the sheet by spotting lL of the radioactive PPi solution (2000 dpm) on a lane in which a mixture of AMP, ADP and ATP was resolved The autoradiographic image was recorded on a storage phosphor screen (Molecular Dynamics, Sunnyvale, CA, now GE Healthcare) for 5–10 min, and analysed with a Typhoon 9400 (GE Healthcare) phosphoimager using the manufacturer’s software Quantitation of [32P]ATP was carried out with imagequant 5.2 (GE Healthcare) software Phosphoimager counts were converted into dpm using a calibration curve made for each TLC sheet by spotting aliquots of [32P]PPi solutions of known radioactivity onto a free lane of the sheet before data collection Limited proteolysis Ec-GluRS or Mt-GluRS (1 mgỈmL)1) were incubated with 0.1–10% (w ⁄ w) Na-tosyl-l-phenyl chloromethyl ketonetreated trypsin (Sigma) in 50 mm Hepes ⁄ NaOH buffer, pH 8.0, at 25 °C, in a final volume of 100–200 lL Before trypsin addition, and at different incubation times, 10 lL aliquots of the reaction mixture were transferred into eppendorf tubes containing 20 lL 2· SDS sample buffer, Na-tosyl-l-lysyl chloromethyl ketone, 20 mm, 10 lL) Proteins were immediately denatured by incubation at 100 °C for 10 This procedure was found to be sufficient to block proteolysis Similar experiments were carried out using Na-tosyl-l-lysyl chloromethyl ketone-treated chymotrypsin instead of trypsin, except for the fact that the reaction was blocked by transferring the 10 lL aliquots in eppendorf tubes containing Na-tosyl-l-phenyl chloromethyl ketone instead of Na-tosyl-l-lysyl chloromethyl ketone Proteolysis products were resolved by SDS ⁄ PAGE, visualized by Coomassie Brilliant Blue staining Their mass was calculated from a calibration curve built with the 14– 202 kDa molecular mass protein standard mix (Sigma) To identify the sites of proteolysis, the proteins were blotted onto Immobilon PSQ (Millipore) membranes after SDS ⁄ PAGE, and subjected to N-terminal sequencing Sequences were compared with the known sequences of FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1413 M tuberculosis glutamyl–tRNA synthetase S Paravisi et al Mt-GluRS (UniProtKB accession number P0A636) and Ec-GluRS (UniProtKB accession number P04805) Small angle X-ray scattering data collection and modeling Synchrotron X-ray scattering data from solutions of Mt- and Ec-GluRs in the presence or absence of ligands were collected at the X33 beamline (DESY, Hamburg, Germany) [49] at protein concentrations (c) ranging from 11 to 0.5 mgỈmL)1 At a sample-detector distance of 2.7 m, the range of momentum transfer 0.1 < s < nm)1 was covered [s = 4p sin(h) ⁄ k, where 2h is the scattering angle and k = 0.15 nm is the X-ray wavelength] The data were processed using standard procedures by the program package primus [27] The forward scattering I(0) and Rg were evaluated using the indirect transform package gnom [50] The effective molecular mass of the solute (MM) was estimated by comparison of the forward scattering I(0) with that from reference solutions of BSA (MM = 66 kDa) The scattering intensities for monomeric and dimeric models of Mt-GluRS were computed by crysol [26] from the atomic coordinates of the discriminating Tt-GluRS (PDB files: 1n75 for the complex with ATP; 1j09 for the complex with ATP and Glu; 1n77 for the complex with tRNA and ATP; 1n78 for the complex with tRNA and GoA; 1g59 for the complex with tRNA) and of the nondiscriminating Te-GluRS (PDB file 2cfo for the complex with Glu) and were used to analyse the oligomeric composition of all the samples The program oligomer [27] was used to find the volume fractions of components minimizing the discrepancy v2 (normalized sum of the reduced standard deviations) between the linear superposition of the weighted intensities of the components and the experimental data from the mixture GluRS ⁄ GluTR interaction Rv0509 encoding the putative Mt-GluTR was amplified from cosmid MTCY20G9 (Institut Pasteur) following a scheme similar to that described for the construction of pETGTS1 The insert of the resulting plasmid (pGTR) was also reamplified in order to remove the stop codon and engineer a XhoI site at the 3¢-end of the ORF to allow for cloning in pET23b digested with NdeI and XhoI The resulting plasmid (pGTRHis) encodes a GluTR species carrying a C-terminal His6 tag (Mt-GluTR–His) With both plasmids the amount of soluble protein increased by co-transforming E coli BL21(DE3) cells with pGTR6 or pGTRHis and p20 [28] The latter plasmid encodes the E coli chaperons DnaJ, DnaK and GrpE Transformed E coli cells were grown in Luria–Bertani medium supplemented with 100 lgỈmL)1 ampicillin, 25 lgỈmL)1 chloramphenicol until the D600 of the culture reached a value of $ The culture was transferred at 15 °C and induction of 1414 the plasmid-encoded proteins was obtained with 0.1 mm IPTG Cells were harvested after 17 h To study the GluTR-GluRS interaction, g of E coli BL21 (DE3, pGTR, p20) cells that had produced the native Mt-GluTR, were resuspended in 20 mm Hepes ⁄ NaOH buffer, pH 8.0, 10% glycerol, mm b-ME and mm phenylmethanesulfonyl fluoride (4 mL) Glass beads (12 g; 0.3 mm diameter) were added and cells were disrupted by applying five cycles of vigorous vortexing (1 min) followed by on ice After twofold dilution, the homogenate and a mL rinse of the glass beads were centrifuged for h at 22 500 g at °C The crude extract (13–15 mL) was diluted fivefold in 20 mm Hepes ⁄ NaOH buffer, pH 8.0, 10% glycerol Mt-His6-GluRS (2 mL of a mgỈmL)1 solution in the same buffer + mm b-ME) was added After incubation for 30 at °C and 10 r.p.m on a rotary shaker, mL of a 50% Ni-nitrilotriacetic acid-Sepharose suspension in the same buffer + mm b-ME was added After h the suspension was poured into a small chromatographic column (inner diameter, 1.6 cm) and the packed resin was extensively washed with the equilibration buffer The column was developed with a stepwise 0–500 mm imidazole gradient in 20 mm Hepes ⁄ NaOH buffer, pH 8.0, mm b-ME Aliquots of the collected fractions were denatured for SDS ⁄ PAGE as described above For each condition two controls were also performed: (a) the His6-GluRS solution was substituted by the same volume of buffer; (b) the cell extract was substituted by a crude extract of E coli BL21 (DE3, pET23b, p20) cells The gels were stained with Coomassie Brilliant Blue, destained and the images were scanned with a ImageScanner (GE Healthcare) GluTR and GluRS proteins in the various fractions were quantified using GluRS and GluTR standards as the reference Bands corresponding to GluTR were identified by western blotting and immunodecoration with rabbit anti-MtGluTR IgG prepared for us by PRIMM srl (Milan) using samples of electrophoretically homogeneous Mt-GluTR– His6 The latter was prepared by Ni-nitrilotriacetic acid– Sepharose affinity chromatograhy under denaturing conditions (6 m urea) from E coli BL21 (DE3) cells transformed with pGTRHis and grown at 25 °C with the addition of IPTG when D600 was and harvested after 15 h A similar experimental scheme was followed by using extracts of cells that had overproduced GluTR–His and purified GluRS Testing the toxicity of Mt-GluRS in E coli BL21(DE3) cells The method described by Baick et al [25] to establish the toxicity of the expression of B subtilis nondiscriminating GluRS in E coli was adapted E coli BL21 (DE3, pETGTS1) cells were grown in Luria–Bertani medium containing 0.1 mgỈmL)1 ampicillin medium at 30 °C and FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS S Paravisi et al 220 r.p.m until the culture reached an D600 of 1.0 Serial dilutions (to 10)5 and · 10)6) were done in Luria– Bertani broth Aliquots (200 lL) of the 10)5 dilution were added to mL 0.7% top agar made with Luria–Bertani medium containing 0.1 mm IPTG or IPTG and 0.1 mgỈmL)1 ampicillin The top agar was poured onto Luria–Bertani plates without ampicillin or with 0.1 mgỈmL)1 ampicillin, respectively Aliquots (200 lL) of the · 10)6 diluted culture were mixed with top agar with or 0.1 mgỈmL)1 ampicillin, and the top agar was poured on Luria–Bertani or Luria–Bertani medium containing 0.1 mgỈmL)1 ampicillin plates, respectively The plates were incubated for up to 48 h at 37 or 25 °C (the latter to mimick large scale growth conditions) The effect of l-Glu or l-Gln (2.5 and 25 mm) in the Luria–Bertani medium was also tested In separate experiments, the toxicity caused by the expression of Mt-GluRS in E coli cells was determined in M9 minimal medium in the presence of different l-Gln or l-Glu concentrations For these experiments E coli BL21 (DE3, pETGTS1) was grown in Luria–Bertani medium containing 0.1 mgỈmL)1 ampicillin serially diluted in 0.9% NaCl to 10)5 and · 10)6 Aliquots (200 lL) were mixed with top agar and then poured on plates as described above except for the fact that M9 medium was used and that three series of samples were prepared containing 0, 2.5 and 25 mm l-Gln or l-Glu in both top agar and plates After incubation at 37 or 25 °C for up to 48 h, the formed colonies were counted Miscellaneous techniques UV–Vis absorbance measurements were done on HP8453 (Agilent Technologies, Santa Clara, CA, USA), Cary219 or Cary100 (Varian, Palo Alto, CA, USA) spectrophotometers connected to water baths Acknowledgements This work was carried out thanks to funds from the Ministero dell’Istruzione, Universita’ e Ricerca MIURPRIN2003 (Rome, Italy), the European Union Contract QLK2-CT-2000-01761 to BC and Fondazione Cariplo (Milano, Italy) Contract 2004-1580 to MAV G Riccardi, E De Rossi and A Aliverti are thanked for the initial cloning of Rv0909 We are grateful to J Lapointe for the gift of pERS, to Dr Rizzi for the gift of decoyinine and of the pyrophosphate analogs tested, to A Mattevi and M Nardini for carrying out crystallization trials, to G Tedeschi and A Negri for performing MALDI-TOF analyses and N-terminal ` sequencing, and to G Deho for helpful discussions PVK, MVP and DIS acknowledge support from the EU design study SAXIER (contract RIDS No 011934) M tuberculosis glutamyl–tRNA synthetase References Jain A & Mondal R (2008) Extensively drug-resistant tuberculosis: current challenges and threats FEMS Immunol Med Microbiol 53, 145–150 Ibba M & Soll D (2000) Aminoacyl–tRNA synthesis Annu Rev Biochem 69, 617–650 Schimmel 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material is available: Fig S1 Mt-GluRS production and purification Fig S2 tRNA aminoacylation activity of Mt-GluRS Fig S3 Determination of the steady-state kinetic parameters of the Mt-GluRS reaction M tuberculosis glutamyl–tRNA synthetase Fig S4 Inhibition of Mt-GluRS by GoA and pyrophosphate Fig S5 Chromatographic separation of Mt-GluRS reaction components Fig S6 PPi ⁄ ATP exchange reaction of Mt-GluRS Fig S7 Alignment of selected GluRS sequences and identification of the limited chymotryptic cleavage sites Fig S8 Analysis of the kinetics of proteolysis of Mt- and Ec-GluRS Fig S9 Minimal models of the proteolytic events leading to fragments M1–M5 of Mt-GluRS and E1-E3 of Ec-GluRS Fig S10 DLS analysis of GluRS aggregation state Fig S11 Spectral properties of Mt-GluTR isolated by using immobilized His6–GluRS and aggregation state Table S1 Alternate substrates and inhibitors of MtGluRS: l-Glu, l-Gln and 2-oxoglutarate Table S2 Alternate substrates and inhibitors of MtGluRS: ATP, AMP, pyrophosphate and their analogs Table S3 Effect of metal ions on the Mt-GluRS activity Table S4 Testing the toxicity of Mt-GluRS in E coli BL21(DE3) cells Table S5 Summary of the properties of the fragments obtained during limited chymotryptic cleavage of Mt- and Ec-GluRS as deduced from the gels shown in Fig 5, main text Table S6 Summary of molecular parameters deduced by SAXS This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 1398–1417 ª 2009 The Authors Journal compilation ª 2009 FEBS 1417 ... the discriminating behaviour of Mt-GluRS in E coli Studies on Tt-GluRS indicated that discriminating and nondiscriminating GluRS can be distinguished on the basis of the presence of a specific... measuring the initial velocity of reactions that contained fixed levels of the inhibitor (I), varying concentration of one of the substrates and constant concentration of others After inspection of. .. activity of Mt-GluRS Fig S3 Determination of the steady-state kinetic parameters of the Mt-GluRS reaction M tuberculosis glutamyl–tRNA synthetase Fig S4 Inhibition of Mt-GluRS by GoA and pyrophosphate