Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis

Hershberger

Chakrabarty

1997

Molecular Microbiology

SummaryWe report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. Immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P 70 , P 65 , P 60 , and P 50 , respectively) of M r 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M r 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P 70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.

Section: Discussionmentioning

confidence: 99%

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

Hershberger

Chakrabarty

1997

Molecular Microbiology

“…Among the seven bacterial proteins tested, two proteins, Pra from P. aeruginosa (Chopade et al, 1997) and P 65 from M. smegmatis , specifically altered the NTP-synthesizing activity of either human or yeast Ndk to GTP or UTP (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Mammalian heterotrimeric G‐protein‐like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Kapatral

Chakrabarty

1997

Molecular Microbiology

SummaryMammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the ␣-, ␤-or the ␥-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk-G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian ␣ and ␤ G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the ␣-, ␤-and ␥-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the ␣-and ␥-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.

“…There is a homolog of Pra in E. coli, known as Era, which is known to be an essential protein, but its exact function is unknown (11,12). We have reported that the E. coli Era protein, having intrinsic GTPase activity, can also form complexes with P. aeruginosa 12-kDa Ndk to modulate its NTP synthesis to GTP (8). To see if other Ndk-binding proteins could also be present in P. aeruginosa, we cross-linked Ndk to Sepharose 4B and allowed the Sepharose-bound Ndk to interact with proteins present in the cell lysates of P. aeruginosa.…”

Section: Elution Of P Aeruginosa Ef-tu From a Ndk-sepharose 4bmentioning

confidence: 99%

“…One is Pk, a 60-kDa protein (5), whereas the other is a 47-kDa Ras-like protein, Pra, with GTPase activity (8). There is a homolog of Pra in E. coli, known as Era, which is known to be an essential protein, but its exact function is unknown (11,12).…”

Section: Elution Of P Aeruginosa Ef-tu From a Ndk-sepharose 4bmentioning

confidence: 99%

“…Though the role of GTP binding proteins has been well documented in eukaryotic cells, the significance of these proteins in prokaryotes remains to be deciphered. Because the enzyme Ndk has been implicated in generating GTP through complex formation with Pk (5) and has also been reported to form complexes with a P. aeruginosa Ras-like GTPbinding protein Pra (8), it was of interest to determine if other GTP-binding proteins that require GTP for their cellular function may also form complexes with Ndk. In this work we demonstrate that EF-Tu forms a complex with both the intact 16-kDa form as well as the truncated 12-kDa form of Ndk to synthesize GTP.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Complex Formation of the Elongation Factor Tu from Pseudomonas aeruginosa with Nucleoside Diphosphate Kinase Modulates Ribosomal GTP Synthesis and Peptide Chain Elongation

Mukhopadhyay

Journal of Biological Chemistry

Walden

et al. 1997

The elongation factor Tu (EF-Tu) from Pseudomonas aeruginosa was purified as a 45-kDa polypeptide that forms a complex with both the 12-and 16-kDa forms of nucleoside-diphosphate kinase (Ndk) and predominantly synthesizes GTP. 70 S ribosomes of P. aeruginosa predominantly synthesize GTP, which is inhibited in presence of anti-Ndk antibodies. Anti-EF-Tu antibodies change the specificity of ribosomal GTP synthesis to all nucleoside triphosphate synthesis. Ndk has been shown to be a part of 30 S ribosomes, whereas EF-Tu is found to be associated with the 50 S ribosomal subunit. These data indicate that GTP synthesis in the ribosome is modulated both by Ndk and by EF-Tu. Peptide chain elongation as measured by polymerization of Phe-tRNA on a poly(U) template in presence of GDP can be inhibited by anti-Ndk antibodies and restored by the addition of GTP. Anti-EF-Tu antibodies similarly inhibit peptide chain elongation by P. aeruginosa ribosomes in the in vitro translation assay; however, this inhibition cannot be overcome by adding back GTP. Because the purified EF-Tu⅐16-kDa Ndk complex predominantly synthesizes GTP, it seems likely that this complex is a significant source of GTP for translational elongation in protein biosynthesis.