{'fraction_non_alphanumeric': 0.035072336694432264, 'fraction_numerical': 0.00657606313020605, 'mean_word_length': 5.269230769230769, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl–tRNA synthetase and determination of its oligomeric structure in solution

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 μm) and ATP (78 μm), 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 μm 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 glutamol‐AMP. 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 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.