Authors: Ling Zhang, Ying-Hui Wang, Xing Zhang...

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

Topics: Anti-Bacterial Agents; Crystallography, X-Ray; Escherichia coli; Models, Molecular; Molecular Conformation; Protein Binding; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; RNA, Transfer; Ribosomal Proteins; Ribosomes; Viomycin

PubMed: 32341159
DOI: 10.1073/pnas.2002888117

Authors: Mikael Holm, Chandra Sekhar Mandava, Måns Ehrenberg...

Applying pre-steady state kinetics to an based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.

Topics: Anti-Bacterial Agents; Cell-Free System; Protein Biosynthesis; Protein Synthesis Inhibitors; Viomycin

PubMed: 31172942
DOI: 10.7554/eLife.46124

Authors: Mikael Holm, Anneli Borg, Måns Ehrenberg...

Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼ 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

Topics: Anti-Bacterial Agents; Bacteria; Dose-Response Relationship, Drug; Guanosine Triphosphate; Peptide Elongation Factor G; Probability; Protein Biosynthesis; Ribosomes; Viomycin

PubMed: 26755601
DOI: 10.1073/pnas.1517541113

Authors: Kristjan Bloudoff, T Martin Schmeing

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

Authors: Rashid Akbergenov, Dmitri Shcherbakov, Tanja Matt...

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

Authors: John J Barkei, Brian M Kevany, Elizabeth A Felnagle...

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

Authors: Robin E Stanley, Gregor Blaha, Robert L Grodzicki...

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

Authors: Elizabeth A Felnagle, Angela M Podevels, John J Barkei...

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

Authors: Xiaobo Fei, Xihou Yin, Ling Zhang...

The nonproteinogenic amino acid capreomycidine is the signature residue found in the tuberactinomycin family of antitubercular peptide antibiotics and an important element of the pharmacophore. Recombinant VioG, a single-module peptide synthetase from the viomycin gene cluster cloned from Streptomyces vinaceus (ATCC11861), specifically activates capreomycidine for incorporation into viomycin (tuberactinomycin B). Insertional disruption of the putative hydroxylase gene vioQ resulted in a mutant that accumulated tuberactinomycin O, suggesting that hydroxylation at C-5 of the capreomycidine residue is a post-assembly event. The inactivated chromosomal copy of vioQ could be complemented with a wild-type copy of the gene to restore viomycin production.

Topics: Anti-Bacterial Agents; Base Sequence; Escherichia coli; Genes, Bacterial; Molecular Structure; Peptide Synthases; Streptomyces; Viomycin

PubMed: 17302456
DOI: 10.1021/np060605u

Authors: Verena Helmetag, Stefan A Samel, Michael G Thomas...

The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)-dependent and alpha-ketoglutarate-dependent Cbeta-hydroxylation of L-arginine during the biosynthesis of the tuberactinomycin antibiotic viomycin. Crystal structures of VioC were determined in complexes with the cofactor Fe(II), the substrate L-arginine, the product (2S,3S)-hydroxyarginine and the coproduct succinate at 1.1-1.3 A resolution. The overall structure reveals a beta-helix core fold with two additional helical subdomains that are common to nonheme iron oxygenases of the clavaminic acid synthase-like superfamily. In contrast to other clavaminic acid synthase-like oxygenases, which catalyze the formation of threo diastereomers, VioC produces the erythro diastereomer of Cbeta-hydroxylated L-arginine. This unexpected stereospecificity is caused by conformational control of the bound substrate, which enforces a gauche(-) conformer for chi(1) instead of the trans conformers observed for the asparagine oxygenase AsnO and other members of the clavaminic acid synthase-like superfamily. Additionally, the substrate specificity of VioC was investigated. The side chain of the L-arginine substrate projects outwards from the active site by undergoing interactions mainly with the C-terminal helical subdomain. Accordingly, VioC exerts broadened substrate specificity by accepting the analogs L-homoarginine and L-canavanine for Cbeta-hydroxylation.

Topics: Anti-Bacterial Agents; Arginine; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Molecular Structure; Nonheme Iron Proteins; Oxygenases; Protein Structure, Secondary; Protein Structure, Tertiary; Stereoisomerism; Streptomyces; Substrate Specificity; Viomycin

PubMed: 19490124
DOI: 10.1111/j.1742-4658.2009.07085.x