부산대학교 도서관

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 forl-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 andl-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 withl-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. [ABSTRACT FROM AUTHOR]