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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 -
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 -
RNA (New York, N.Y.) Feb 2012The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after...
The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.
Topics: Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; GTP Phosphohydrolases; Guanosine Triphosphate; Models, Molecular; Peptide Elongation Factor G; Peptide Termination Factors; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; RNA, Ribosomal, 23S; Ribosomes; Translocation, Genetic; Viomycin
PubMed: 22187675
DOI: 10.1261/rna.031187.111 -
Antimicrobial Agents and Chemotherapy Oct 2011Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including...
Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.
Topics: Aminoglycosides; Antitubercular Agents; Bacterial Proteins; Capreomycin; Drug Resistance, Multiple, Bacterial; Enviomycin; Mutagenesis, Site-Directed; Mycobacterium tuberculosis; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribosomes; Viomycin
PubMed: 21768509
DOI: 10.1128/AAC.00628-11 -
Chembiochem : a European Journal of... Aug 2011
Topics: Antitubercular Agents; Bacterial Proteins; Capreomycin; Chromatography, High Pressure Liquid; DNA Primers; Escherichia coli; Extensively Drug-Resistant Tuberculosis; Metabolic Engineering; Mycobacterium tuberculosis; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Plasmids; Recombinant Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptomyces lividans; Structural Homology, Protein; Transformation, Genetic; Viomycin
PubMed: 21739558
DOI: 10.1002/cbic.201100193 -
Biophysical Journal May 2011Transfer RNA (tRNA) translocates inside the ribosome during translation. We studied the interaction strengths between the ribosome and tRNA at various stages of...
Transfer RNA (tRNA) translocates inside the ribosome during translation. We studied the interaction strengths between the ribosome and tRNA at various stages of translocation. We utilized an optical trap to measure the mechanical force to rupture tRNA from the ribosome. We measured the rupture forces of aminoacyl tRNA or peptidyl tRNA mimic from the ribosome in a prepeptidyl transfer state, the pretranslocational state, and the posttranslocational state. In addition, we measured the interaction strength between the ribosome and aminoacyl-tRNA in presence of viomycin. Based on the interaction strengths between the ribosome and tRNA under these conditions, 1), we concluded that tRNA interaction with the 30S subunit is far more important than the interaction with the 50S subunit in the mechanism of translocation; and 2), we propose a mechanism of translocation where the ribosomal ratchet motion, with the aid of EF-G, drives tRNA translocation.
Topics: Escherichia coli; Peptide Chain Elongation, Translational; Protein Biosynthesis; RNA, Transfer; RNA, Transfer, Amino Acyl; Ribosomes
PubMed: 21539788
DOI: 10.1016/j.bpj.2011.03.023 -
Biochemistry Oct 2010The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of...
The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.
Topics: Bacteria; Bacterial Proteins; Capreomycin; Multigene Family; Peptide Synthases; Viomycin
PubMed: 20845982
DOI: 10.1021/bi1012854 -
Journal of Clinical Microbiology May 2010The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible...
The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.
Topics: Aminoglycosides; Antitubercular Agents; Culture Media; Humans; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Peptides, Cyclic; Tuberculosis
PubMed: 20237102
DOI: 10.1128/JCM.00240-10 -
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 -
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