Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

We overexpressed M. tuberculosis Obg in Escherichia coli and then purified the protein. This protein binds to, hydrolyzes and is phosphorylated with GTP. An anti-Obg antiserum, raised against the purified Obg, detects a 55 kDa protein in immunoblots of M. tuberculosis extracts. Immunoblotting also discloses that cultured M. tuberculosis cells contain increased amounts of Obg in the late log phase and in the stationary phase. Obg is also associated with ribosomes in M. tuberculosis, and it is distributed to all three ribosomal fractions (30 S, 50 S and 70 S). Finally, yeast two-hybrid analysis reveals that Obg interacts with the stress protein UsfX, indicating that M. tuberculosis Obg, like other bacterial Obgs, is a stress related protein.Although its GTP-hydrolyzing and phosphorylating activities resemble those of other bacterial Obg homologues, M. tuberculosis Obg differs from them in these respects: (a) preferential association with the bacterial membrane; (b) association with all three ribosomal subunits, and (c) binding to the stress protein UsfX, rather than to RelA. Generation of mutant alleles of Obg of M. tuberculosis, and their characterization in vivo, may provide additional insights regarding its role in this important human pathogen.GTP-binding proteins are found in all living organisms, and they play critical roles in fundamental processes such as cell proliferation, development, signal transduction and protein translation [1,2]. In general, these proteins are hydrolase enzymes that convert GTP into GDP, allowing transfer of the GTP terminal phosphate group to a target protein. As a consequence of this transfer, the highly conserved domains (G1, G2, G3, G4 and G5) of GTP-binding proteins undergo conformational changes that are detected by downstream effector proteins [3,4], leading to specific outcomes.Comparison of bacterial genomes, across all taxa, has shown that at least eleven highly conserved GTP-binding proteins are present in prokaryotes [5]. A