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RNA (New York, N.Y.) Sep 2021Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the...
Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.
Topics: Anti-Bacterial Agents; Biological Transport; Cinnamates; Escherichia coli; Hygromycin B; Kanamycin; Kinetics; Neomycin; Paromomycin; Peptide Elongation Factor G; Protein Biosynthesis; RNA, Messenger; RNA, Transfer; Ribosome Subunits; Spectinomycin; Streptomycin; Viomycin
PubMed: 34117118
DOI: 10.1261/rna.078758.121 -
Molecular Microbiology Sep 2012The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and...
The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2'-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to capreomycin and viomycin. We report here that TlyA orthologues occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyA(I) , has shorter N- and C-termini and methylates only C1920; the second group (now TlyA(II) ) includes the mycobacterial enzyme, and these longer orthologues methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologues. Amino acid substitutions at the N-terminus of TlyA(II) reduce its ability to methylate these substrates. Growing pairs of recombinant TlyA(II) Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of capreomycin. The findings reveal that 2'-O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of capreomycin and viomycin on the bacterial ribosome.
Topics: Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Capreomycin; Methylation; Microbial Sensitivity Tests; Models, Molecular; Nucleic Acid Conformation; RNA, Ribosomal; Ribosome Subunits; Viomycin; tRNA Methyltransferases
PubMed: 22779429
DOI: 10.1111/j.1365-2958.2012.08168.x -
Investigations into viomycin biosynthesis by using heterologous production in Streptomyces lividans.Chembiochem : a European Journal of... Jan 2009Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is...
Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted-gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with beta-lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the beta-amino group of L-2,3-diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and capreomycin.
Topics: Amino Acids; Antitubercular Agents; Bacterial Proteins; Gene Deletion; Multigene Family; Streptomyces lividans; Viomycin
PubMed: 19105177
DOI: 10.1002/cbic.200800646 -
Proceedings of the National Academy of... Apr 2022Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are...
Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2′-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.
Topics: Bacterial Proteins; Catalytic Domain; Drug Resistance, Bacterial; Methyltransferases; Mycobacterium tuberculosis; Protein Conformation, alpha-Helical; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribosome Subunits, Large, Bacterial
PubMed: 35357969
DOI: 10.1073/pnas.2120352119 -
Nucleic Acids Research Sep 2017Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to...
Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine-Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon.
Topics: Bacterial Proteins; Codon, Terminator; Escherichia coli; Fluorescence Polarization; Fusidic Acid; Guanosine Triphosphate; Kinetics; Models, Biological; Peptide Elongation Factor G; Prokaryotic Initiation Factor-3; RNA, Bacterial; RNA, Messenger; RNA, Transfer; Ribosome Subunits; Ribosomes; Thiostrepton; Viomycin
PubMed: 28973468
DOI: 10.1093/nar/gkx694 -
Journal of Biomolecular Structure &... Feb 20223CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular...
3CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular dynamics simulation, we found 23 approved clinical drugs such as Viomycin, Capastat, Carfilzomib and Saquinavir, which showed high affinity with the 3CL active sites. These findings showed that there were potential drugs that inhibit SARS-Cov-2's 3CL in the current clinical drug library, and these drugs can be further tested or chemically modified for the treatment of COVID-19.Communicated by Ramaswamy H. Sarma.
Topics: COVID-19; Humans; Molecular Docking Simulation; Peptide Hydrolases; Pharmaceutical Preparations; Protease Inhibitors; SARS-CoV-2
PubMed: 32909528
DOI: 10.1080/07391102.2020.1817786 -
Acta Crystallographica. Section F,... Apr 2013Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize important secondary metabolites such as antibiotics. NRPSs follow a modular...
Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize important secondary metabolites such as antibiotics. NRPSs follow a modular synthetic logic whereby each successive amino-acid monomer is added to the peptide chain by successive multi-domain modules. The condensation domain catalyzes the central chemical event in the synthetic cycle, peptide-bond formation, and is present in every elongation module of the NRPS. Viomycin is an antituberculosis nonribosomal peptide that is synthesized by a series of four NRPS proteins and then modified by tailoring proteins. In order to study the mechanisms of peptide-bond formation in viomycin and in NRPSs in general, a structural study of the first condensation domain of the viomycin synthetase protein VioA (VioA-C1) was initiated. The gene for VioA-C1 was cloned from genomic DNA of Streptomyces vinaceus, expressed as an octahistidine-tagged construct and purified by column chromatography. VioA-C1 was crystallized using the sitting-drop vapor-diffusion method. X-ray diffraction data were collected on a rotating-anode source to 2.9 Å resolution. The data could be indexed in the orthorhombic space group P212121, with unit-cell parameters a = 46.165, b = 68.335, c = 146.423 Å. There is likely to be one monomer in the asymmetric unit, giving a solvent content of 49.2% and a Matthews coefficient (VM) of 2.42 Å(3) Da(-1). Structural determination is in progress.
Topics: Crystallization; Crystallography, X-Ray; Peptide Synthases; Streptomyces; Viomycin
PubMed: 23545648
DOI: 10.1107/S1744309113004004 -
Nature Structural & Molecular Biology Mar 2010Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis....
Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-A resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.
Topics: Antitubercular Agents; Capreomycin; Crystallography, X-Ray; Molecular Sequence Data; Molecular Structure; Protein Binding; Protein Structure, Secondary; RNA, Transfer; Ribosomes; Thermus thermophilus; Viomycin
PubMed: 20154709
DOI: 10.1038/nsmb.1755 -
Antimicrobial Agents and Chemotherapy Aug 2005Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them...
Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.
Topics: Amikacin; Antibiotics, Antitubercular; Capreomycin; Drug Resistance, Bacterial; Genes, rRNA; Humans; Kanamycin; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis; Protein Biosynthesis; RNA, Ribosomal, 16S; Viomycin
PubMed: 16048924
DOI: 10.1128/AAC.49.8.3192-3197.2005 -
Journal of Molecular Biology Mar 2010EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain...
EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST-->I(1)-->I(2)-->I(3)-->PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3'-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I(3) rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I(3) is likely to be important for optimizing functional protein biosynthesis.
Topics: Base Sequence; Catalysis; Escherichia coli; Escherichia coli Proteins; Kinetics; Models, Biological; Peptide Initiation Factors; Puromycin; RNA, Bacterial; RNA, Messenger; RNA, Transfer, Amino Acyl; Ribosomes; Spectrometry, Fluorescence; Transcriptional Elongation Factors
PubMed: 20045415
DOI: 10.1016/j.jmb.2009.12.043