guanosine-diphosphate and pulvomycin

The effect of pulvomycin on the biochemical and fluorescence spectroscopic properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α), the functional analog of eubacterial EF-Tu, was investigated. The antibiotic was able to reduce in vitro the rate of protein synthesis however, the concentration of pulvomycin leading to 50% inhibition (173 μM) was two order of magnitude higher but one order lower than that required in eubacteria and eukarya, respectively. The effect of the antibiotic on the partial reactions catalysed by SsEF-1α indicated that pulvomycin was able to decrease the affinity of the elongation factor toward aa-tRNA only in the presence of GTP, to an extent similar to that measured in the presence of GDP. Moreover, the antibiotic produced an increase of the intrinsic GTPase catalysed by SsEF-1α, but not that of its engineered forms. Finally, pulvomycin induced a variation in fluorescence spectrum of the aromatic region of the elongation factor and its truncated forms. These spectroscopic results suggested that a conformational change of the elongation factor takes place upon interaction with the antibiotic. This finding was confirmed by the protection against chemical denaturation of SsEF-1α, observed in the presence of pulvomycin. However, a stabilising effect of the antibiotic directly on the protein in the complex could takes place.

Topics: Aminoglycosides; Archaeal Proteins; Bacteria; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Peptide Elongation Factor 1; Protein Binding; Protein Denaturation; Protein Synthesis Inhibitors; Recombinant Proteins; RNA, Transfer, Amino Acyl; Spectrometry, Fluorescence; Sulfolobus solfataricus; Thermodynamics

The antibiotic pulvomycin is an inhibitor of protein synthesis that prevents the formation of the ternary complex between elongation factor (EF-) Tu.GTP and aminoacyl-tRNA. In this report, novel aspects of its action on EF-Tu are described. Pulvomycin markedly affects the equilibrium and kinetics of the EF-Tu-nucleotide interaction, particularly of the EF-Tu.GTP complex. The binding affinity of EF-Tu for GTP is increased 1000 times, mainly as the consequence of a dramatic decrease in the dissociation rate of this complex. In contrast, the affinity for GDP is decreased 10-fold due to a marked increase in the dissociation rate of EF-Tu.GDP (25-fold) that mimics the action of EF-Ts, the GDP/GTP exchange factor of EF-Tu. The effects of pulvomycin and EF-Ts can coexist and are simply additive, supporting the conclusion that these two ligands interact with different sites of EF-Tu. This is further confirmed on native PAGE by the ability of EF-Tu to bind the EF-Ts and the antibiotic simultaneously. Pulvomycin enhances the intrinsic EF-Tu GTPase activity, like kirromycin, though to a much more modest extent. As with kirromycin, this stimulation depends on the concentration and nature of the monovalent cations, Li(+) being the most effective one, followed by Na(+), K(+), and NH(4)(+). In the presence of pulvomycin (in contrast to kirromycin), aa-tRNA and/or ribosomes do not enhance the GTPase activity of EF-Tu. The property of pulvomycin to modify selectively the conformation(s) of EF-Tu is also supported by its effect on heat- and urea-dependent denaturation, and tryptic digestion of the protein. Specific differences and similarities between the action of pulvomycin and the other EF-Tu-specific antibiotics are described and discussed.

Topics: Aminoglycosides; Anti-Bacterial Agents; Binding Sites; Enzyme Inhibitors; Enzyme Stability; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Peptide Elongation Factor Tu; Peptide Elongation Factors; Peptides, Cyclic; Protein Denaturation; Pyridones; Thiazoles; Trypsin; Urea

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

Topics: Actinomycetales; Adenine; Amino Acid Substitution; Aminoglycosides; Anti-Bacterial Agents; Drug Resistance, Microbial; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Models, Molecular; Mutation; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Poly U; Protein Binding; Protein Biosynthesis; Protein Conformation; RNA, Bacterial; RNA, Transfer, Amino Acyl; Thermodynamics; Thermus; Thiazoles

Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP). Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two mutants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin. Moreover, the mutant [R230V, R233F]EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.

Topics: Aminoglycosides; Anti-Bacterial Agents; Base Sequence; DNA Primers; DNA, Bacterial; Drug Resistance, Microbial; Escherichia coli; Genes, Bacterial; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Pyridones

The sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Microbial; Gram-Negative Bacteria; Gram-Positive Bacteria; Guanosine Diphosphate; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Pyridones; Species Specificity; Thiazoles

In a previous study (C. C. Hall, J. D. Watkins, and N. H. Georgopapadakou, Antimicrob. Agents Chemother. 33:322-325, 1989), the elongation factor Tu (EF-Tu) from Staphylococcus aureus was found to be insensitive to a series of kirromycin analogs which were inhibitory to the EF-Tu from Escherichia coli. In the present study, the EF-Tu from S. aureus was partially purified and characterized. Its apparent molecular mass was approximately 41,000 Da, and the enzyme copurified with EF-Ts (molecular mass, 34,000 Da). S. aureus EF-Tu differed from its E. coli counterpart in that it bound negligible amounts of [3H]GDP, in addition to being insensitive to pulvomycin and aurodox (50% inhibitory concentrations, approximately 100 and 1,000 microM, respectively, versus 2 and 0.2 microM, respectively, for E. coli). The results are consistent with the formation of a stable EF-Tu.EF-Ts complex that affects the interaction of EF-Tu with guanine nucleotides and inhibitors.

Topics: Adenosine Triphosphatases; Aminoglycosides; Anti-Bacterial Agents; Aurodox; Chromatography, DEAE-Cellulose; Drug Resistance, Microbial; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Molecular Weight; Peptide Biosynthesis; Peptide Elongation Factor Tu; Peptides; Pyridones; Staphylococcus aureus

The new thiazolyl peptide antibiotic GE2270 A, isolated from Planobispora rosea strain ATCC 53773, is shown to inhibit bacterial protein biosynthesis in vitro by affecting specifically the GTP-bound form of elongation factor Tu (EF-Tu). The 'off' rate of EF-Tu.GTP is slowed down 400-fold, locking GTP on EF-Tu, whereas EF-Tu.GDP is unaffected. Therefore, on the EF-Tu.guanine nucleotide interaction, GE2270 A mimicks the effect of aa-tRNA. In line with this, the binding of aa-tRNA to EF-Tu.GTP is hindered by the antibiotic, as shown by the absence of a stable ternary complex and the inhibition of the enzymatic binding of aa-tRNA to the ribosome. This blocks the elongation cycle. GE2270 A does not essentially modify the intrinsic GTPase activity of EF-Tu, but impairs the stimulation by ribosomes of this reaction. The negative effect of GE2270 A on the EF-Tu.GTP interaction with aa-tRNA bears similarities with that of the structurally unrelated pulvomycin, whereas marked differences were found by comparing the effects of these two antibiotics on EF-Tu.GDP. This work emphasizes the varieties of the transitional conformations which tune the EF-Tu interaction with GTP and GDP.

Topics: Aminoglycosides; Anti-Bacterial Agents; Escherichia coli; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Molecular Structure; Peptide Chain Elongation, Translational; Peptide Elongation Factor Tu; Peptides; Peptides, Cyclic; Protein Binding; Ribosomes; Thiazoles