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 tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.7 microm) and ATP (78 microm), 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 mum 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 active. By using affinity chromatography and His(6)-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-tRNA(Glu) toward protein or tetrapyrrole biosynthesis.
Paravisi
Stefano
S
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan, Italy.
Fumagalli
Gianluca
G
Riva
Milena
M
Morandi
Paola
P
Morosi
Rachele
R
Konarev
Peter V
PV
Petoukhov
Maxim V
MV
Bernier
Stéphane
S
Chênevert
Robert
R
Svergun
Dmitri I
DI
Curti
Bruno
B
Vanoni
Maria A
MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005975:Glutamate-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism; D012996:Solutions
D001426:Bacterial Proteins; D012346:RNA, Transfer, Amino Acyl; D012996:Solutions; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D005975:Glutamate-tRNA Ligase