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Michael G Thomas - One of the best experts on this subject based on the ideXlab platform.
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mechanistically distinct nonribosomal peptide synthetases assemble the structurally related antibiotics Viomycin and capreomycin
ChemBioChem, 2011Co-Authors: John J Barkei, Elizabeth A Felnagle, Angela M Podevels, Michael G ThomasAbstract:Tuberculosis (TB) is a worldwide burden for human health. More than 1.8 million people succumbed to TB infection in 2008 alone, and as many as two billion people may be passively infected by Mycobacterium tuberculosis, the causative agent of TB.[1] One of the challenges facing the successful treatment of M. tuberculosis infections is the development of strains that are resistant to many of the antibiotics used in the clinic. Cases of multidrug-resistant TB (MDR-TB), defined as TB caused by a M. tuberculosis strain that is resistant to both rifampin and isoniazid, have been identified in nearly every country surveyed.[1, 2] Extensively drug-resistant TB (XDR-TB), defined as MDR-TB that is also resistant to a flouroquinolone and at least one of three injectables (capreomycin, streptomycin, or amikacin), has spread to more than 45 countries, including the United States.[1, 2] The development and spread of drug-resistant strains of M. tuberculosis has put a high priority on the development of new antituberculosis drugs and the generation of derivatives of known drugs that regain their antibiotic activity against resistant strains. The tuberactinomycin family of antituberculosis drugs are important components of our drug arsenal against drug-resistant M. tuberculosis. The most prominent member of this family is capreomycin (CMN), which is a key drug in the treatment of MDR-TB based on its inclusion on the World Health Organization’s “Model List of Essential Medicines.”[3] Furthermore, if CMN is the injectable that XDR-TB infection is resistant to, it almost guarantees treatment failure.[4] Based on its level of importance in treating drug-resistant forms of TB, it is important that new CMN derivatives be developed that regain activity against resistant M. tuberculosis strains. Synthetic procedures to synthesize derivatives have been difficult.[5, 6] More success has been accomplished by semisynthetic approaches,[7–9] but the derivatives are limited by the functional groups present on the cyclic pentapeptide core. Complementing these approaches with metabolic engineering of the enzymology that produces the cyclic pentapeptide core has the potential of enabling further structural diversification. Our focus is on the metabolic engineering aspect of drug development. Harnessing the full potential of metabolic engineering to generate new drug derivatives requires a complete understanding of how the targeted drug is biosynthesized by the producing organism. To this end, we have focused on understanding tuberactinomycin biosynthesis at the molecular and biochemical level by using CMN and the structural analog Viomycin (VIO) as model systems. Our focus on both CMN and VIO was based on our hypothesis that by studying the biosynthesis of two structurally related molecules (Scheme 1), we would gain considerable insights into how the tuberactinomycins are biosynthesized and structurally modified by the natural enzymology. To date, we have sequenced and annotated the CMN and VIO biosynthesis gene clusters,[10, 11] reconstituted CMN and VIO production in the heterologous host Streptomyces lividans,[12] biochemically characterized L-capreomycidine formation,[13] and identified the amino acids activated by each of the adenylation (A) domains of the CMN nonribosomal peptide synthetases (NRPS) and some of the A domains of the VIO NRPS (Scheme 2).[14] Zabriskie and colleagues have also contributed important genetic and biochemical information that enable more refined models of tuberactinomycin biosynthesis to be developed.[15–18] Open in a separate window Scheme 1 Chemical structures of Viomycin (VIO), tuberactinamine A (TMN A; des-β-lysine VIO) and the four derivatives that make up capreomycin (CMN). The numbering within the cyclic pentapeptide cores of the antibiotics is used to identify positions noted in the text.
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structural basis for the erythro stereospecificity of the l arginine oxygenase vioc in Viomycin biosynthesis
FEBS Journal, 2009Co-Authors: Verena Helmetag, Michael G Thomas, Stefan A Samel, Mohamed A Marahiel, Larsoliver EssenAbstract: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.
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investigations into Viomycin biosynthesis by using heterologous production in streptomyces lividans
ChemBioChem, 2009Co-Authors: John J Barkei, Brian M Kevany, Elizabeth A Felnagle, Michael G ThomasAbstract: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.
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deciphering tuberactinomycin biosynthesis isolation sequencing and annotation of the Viomycin biosynthetic gene cluster
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Michael G Thomas, Yolande A Chan, Sarah G OzanickAbstract:The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, Viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of Viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.
Harry F Noller - One of the best experts on this subject based on the ideXlab platform.
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the structural basis for inhibition of ribosomal translocation by Viomycin
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Ling Zhang, Yinghui Wang, Xing Zhang, Laura Lancaster, Jie Zhou, Harry F NollerAbstract: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 Escherichia coli 70S ribosome complexes trapped in a rotated state by Viomycin. The 3.8-A 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-A 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.
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The antibiotic Viomycin traps the ribosome in an intermediate state of translocation
Nature Structural & Molecular Biology, 2007Co-Authors: Dmitri N Ermolenko, P Clint Spiegel, Zigurts K Majumdar, Robyn P Hickerson, Robert M Clegg, Harry F NollerAbstract:During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that Viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that Viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that Viomycin induces formation of a hybrid-state translocation intermediate. Thus, Viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is Viomycin sensitive, the hybrid state may be present in vivo .
Courtney E Maus - One of the best experts on this subject based on the ideXlab platform.
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capreomycin binds across the ribosomal subunit interface using tlya encoded 2 o methylations in 16s and 23s rrnas
Molecular Cell, 2006Co-Authors: Shanna K Johansen, Courtney E Maus, Bonnie B Plikaytis, Stephen DouthwaiteAbstract:The cyclic peptide antibiotics capreomycin and Viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and Viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and Viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and Viomycin at the ribosome subunit interface.
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molecular analysis of cross resistance to capreomycin kanamycin amikacin and Viomycin in mycobacterium tuberculosis
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: Courtney E Maus, Bonnie B Plikaytis, Thomas M ShinnickAbstract: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.
Elizabeth A Felnagle - One of the best experts on this subject based on the ideXlab platform.
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mechanistically distinct nonribosomal peptide synthetases assemble the structurally related antibiotics Viomycin and capreomycin
ChemBioChem, 2011Co-Authors: John J Barkei, Elizabeth A Felnagle, Angela M Podevels, Michael G ThomasAbstract:Tuberculosis (TB) is a worldwide burden for human health. More than 1.8 million people succumbed to TB infection in 2008 alone, and as many as two billion people may be passively infected by Mycobacterium tuberculosis, the causative agent of TB.[1] One of the challenges facing the successful treatment of M. tuberculosis infections is the development of strains that are resistant to many of the antibiotics used in the clinic. Cases of multidrug-resistant TB (MDR-TB), defined as TB caused by a M. tuberculosis strain that is resistant to both rifampin and isoniazid, have been identified in nearly every country surveyed.[1, 2] Extensively drug-resistant TB (XDR-TB), defined as MDR-TB that is also resistant to a flouroquinolone and at least one of three injectables (capreomycin, streptomycin, or amikacin), has spread to more than 45 countries, including the United States.[1, 2] The development and spread of drug-resistant strains of M. tuberculosis has put a high priority on the development of new antituberculosis drugs and the generation of derivatives of known drugs that regain their antibiotic activity against resistant strains. The tuberactinomycin family of antituberculosis drugs are important components of our drug arsenal against drug-resistant M. tuberculosis. The most prominent member of this family is capreomycin (CMN), which is a key drug in the treatment of MDR-TB based on its inclusion on the World Health Organization’s “Model List of Essential Medicines.”[3] Furthermore, if CMN is the injectable that XDR-TB infection is resistant to, it almost guarantees treatment failure.[4] Based on its level of importance in treating drug-resistant forms of TB, it is important that new CMN derivatives be developed that regain activity against resistant M. tuberculosis strains. Synthetic procedures to synthesize derivatives have been difficult.[5, 6] More success has been accomplished by semisynthetic approaches,[7–9] but the derivatives are limited by the functional groups present on the cyclic pentapeptide core. Complementing these approaches with metabolic engineering of the enzymology that produces the cyclic pentapeptide core has the potential of enabling further structural diversification. Our focus is on the metabolic engineering aspect of drug development. Harnessing the full potential of metabolic engineering to generate new drug derivatives requires a complete understanding of how the targeted drug is biosynthesized by the producing organism. To this end, we have focused on understanding tuberactinomycin biosynthesis at the molecular and biochemical level by using CMN and the structural analog Viomycin (VIO) as model systems. Our focus on both CMN and VIO was based on our hypothesis that by studying the biosynthesis of two structurally related molecules (Scheme 1), we would gain considerable insights into how the tuberactinomycins are biosynthesized and structurally modified by the natural enzymology. To date, we have sequenced and annotated the CMN and VIO biosynthesis gene clusters,[10, 11] reconstituted CMN and VIO production in the heterologous host Streptomyces lividans,[12] biochemically characterized L-capreomycidine formation,[13] and identified the amino acids activated by each of the adenylation (A) domains of the CMN nonribosomal peptide synthetases (NRPS) and some of the A domains of the VIO NRPS (Scheme 2).[14] Zabriskie and colleagues have also contributed important genetic and biochemical information that enable more refined models of tuberactinomycin biosynthesis to be developed.[15–18] Open in a separate window Scheme 1 Chemical structures of Viomycin (VIO), tuberactinamine A (TMN A; des-β-lysine VIO) and the four derivatives that make up capreomycin (CMN). The numbering within the cyclic pentapeptide cores of the antibiotics is used to identify positions noted in the text.
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investigations into Viomycin biosynthesis by using heterologous production in streptomyces lividans
ChemBioChem, 2009Co-Authors: John J Barkei, Brian M Kevany, Elizabeth A Felnagle, Michael G ThomasAbstract: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.
Mikael Holm - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of Viomycin inhibition of peptide elongation in bacteria
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Mikael Holm, Anneli Borg, Mans Ehrenberg, Suparna SanyalAbstract: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.
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A tale of two antibiotics : Fusidic acid and Viomycin
2016Co-Authors: Mikael HolmAbstract:Antibiotics that target the bacterial ribosome make up about half of all clinically used antibiotics. We have studied two ribosome targeting drugs: Fusidic acid and Viomycin. Fusidic acid inhibits ...
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insights into the fidelity mechanism of mrna decoding from characterization of Viomycin induced miscoding in translation
2016Co-Authors: Mikael Holm, Suparna SanyalAbstract:Using pre-steady state kinetics and an E. coli based in vitro translation system we have studied the effect of the antibiotic Viomycin on mRNA decoding. We find that Viomycin binds to the ribosome ...