Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl–tRNA synthetase and determination of its oligomeric structure in solution

Abstract

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 tRNA Glu to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k cat (130 min ) and K M for tRNA (0.7 lm) and ATP (78 lm), but to differ by a one order of magnitude higher K M 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 K i 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 tRNA Gln with l-Glu and that GluRS ⁄ tRNA Gln 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 Abbreviations

Redox status affects the catalytic activity of glutamyl-tRNA synthetase

“…An interaction between GluRS and GluTR has been proposed [29,30]. Our data indicate that a complex between GluRS and GluTR from A. ferrooxidans is also formed (O. Orellana, D. Pezoa, P. Alamos, unpublished results), which could potentially channel Glu-tRNA directly for tetrapyrrole synthesis.…”

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

“…In humans upon phosphorylation, GluProRS is released from the MSC and associates with NS1-associated protein 1, ribosomal protein L13a, and glyceraldehyde-3-phosphate dehydrogenase to silence translation of certain mRNAs related to the inflammatory response (30). In Chlamydomonas reinhardtii and Mycobacterium tuberculosis glutamyl-tRNA reductase (GluTR) binds GluRS likely to divert Glu-tRNA Glu synthesis from use in translation to tetrapyrrole biosynthesis (31,32). Given that many archaea posses GluTR (33), there may be competition for binding with ND-GluRS between GatDE and GluTR.…”

Non-Canonical Heme-Binding Proteins

“…The authors found that heme content in GluTR varied according to the concentration of heme provided in the culture medium, and that heme bound preferentially to the dimeric state of the protein. 92 Variability in heme content for GluTR was reported for two other species, namely C. vibrioforme (1 heme per monomer) 93 and M. tuberculosis (1 heme per 16 monomers) 94 GluTR. Binding of heme was accompanied by the formation of a low-spin complex with a Soret peak maximum appearing at 420 nm.…”