NMR and Molecular Modelling Studies of the Binding of Amicetin Antibiotic to Conserved Secondary Structural Motifs of 23S Ribosomal RNAs

Donarski, James; Shammas, Christos; Banks, Ryan; Ramesh, Vasudevan

doi:10.1038/ja.2006.25

Cited by 14 publications

(6 citation statements)

References 27 publications

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“…Amicetin has been described as a universal antibiotic, exerting effects against microorganisms of different evolutionary origins, including archaea, bacteria, and eukarya (44), by functioning as a peptidyl transferase inhibitor to block protein biosynthesis (41). Although the binding of amicetin to a conserved structural motif of 23S rRNAs has been investigated by using site-directed mutations, chemical footprinting, and nuclear magnetic resonance (NMR) structure and molecular modeling studies (18,44), the exact working mechanism for amicetin remains elusive.…”

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confidence: 99%

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

Zhang

et al. 2012

Appl Environ Microbiol

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ABSTRACTAmicetin, an antibacterial and antiviral agent, belongs to a group of disaccharide nucleoside antibiotics featuring an α-(1→4)-glycoside bond in the disaccharide moiety. In this study, the amicetin biosynthesis gene cluster was cloned fromStreptomyces vinaceusdrappusNRRL 2363 and localized on a 37-kb contiguous DNA region. Heterologous expression of the amicetin biosynthesis gene cluster inStreptomyces lividansTK64 resulted in the production of amicetin and its analogues, thereby confirming the identity of theamigene cluster.In silicosequence analysis revealed that 21 genes were putatively involved in amicetin biosynthesis, including 3 for regulation and transportation, 10 for disaccharide biosynthesis, and 8 for the formation of the amicetin skeleton by the linkage of cytosine,p-aminobenzoic acid (PABA), and the terminal (+)-α-methylserine moieties. The inactivation of the benzoate coenzyme A (benzoate-CoA) ligase geneamiLand theN-acetyltransferase geneamiFled to two mutants that accumulated the same two compounds, cytosamine and 4-acetamido-3-hydroxybenzoic acid. These data indicated that AmiF functioned as an amide synthethase to link cytosine and PABA. The inactivation ofamiR, encoding an acyl-CoA-acyl carrier protein transacylase, resulted in the production of plicacetin and norplicacetin, indicating AmiR to be responsible for attachment of the terminal methylserine moiety to form another amide bond. These findings implicated two alternative strategies for amide bond formation in amicetin biosynthesis.

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confidence: 99%

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

Zhang

et al. 2012

Appl Environ Microbiol

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“…They suggested a rigorous evaluation of cytimidine’s activity would be useful to evaluate their model (as it contains the α-methylserine residue). Perhaps more vexing was the suggestion from NMR experiments that amicetin adopted a highly strained conformation with an s -cis amide at the p -aminobenzoate–cytosine junction (Figure B). , Extensive intramolecular hydrogen bonding between the α-methylserine and disaccharide was suggested to stabilize this high energy conformation. This made it almost impossible to make heads or tails of where to start!…”

Section: The Peptidylcytosine Antibioticsmentioning

confidence: 99%

Dissecting the Nucleoside Antibiotics as Universal Translation Inhibitors

2021

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Metrics & MoreArticle Recommendations CONSPECTUS: Without question, natural products have provided the lion share of leads, if not drugs themselves, for the treatment of bacterial infections. The bacterial arms race, fueled by selection and survival pressures has delivered a natural arsenal of small molecules targeting the most essential of life processes. Antibiotics that target these critical intracellular processes face the formidable defense of both penetrating a bacterial cell membrane and avoiding efflux to exert their effect. These challenges are especially effective in Gramnegative (Gram-(−)) bacteria, which have a double membrane structure and efficient efflux systems from the combination of outer-membrane porins and inner membrane proton pumps. In this landscape of offense and defense, our clinically used antibiotics have only successfully targeted three intracellular processes for therapeutic intervention in Gram-(−) bacteria: dihydrofolate biosynthesis, transcription, and translation. Not surprisingly, such critical survival machinery is a popular target for bacterial warfare, and eight of our 14 classes of commonly used antibiotics target translation with the bacterial ribosome remaining one the most vetted targets for antimicrobial therapy. On the plus side, its anionic character attracts cationic inhibitors, which are generally more capable of penetrating the bacterial cell wall, and clinical resistance rates are usually manageable as mutation of such a highly evolved machine is difficult. On the down side, this highly evolved machine renders it difficult to inhibit selectively, and the inhibition of prokaryotic translation versus both eukaryotic cellular and mitochondrial translation is critical for clinical development and minimization of undesired toxicities.A class of natural products known as the "nucleoside antibiotics" have historically been recognized as universal inhibitors of the ribosome and can inhibit translation in prokaryotes, eukaryotes, and archaea. While they have served an essential role in dissecting the biochemical underpinnings of the enzymatic functions of the ribosome, they have not proven therapeutically useful as they target the highly conserved rRNA in the P-site and are toxic to mammalian cells. In this Account, we describe our studies on the natural product amicetin, a nucleoside antibiotic that we have demonstrated to break the rule of being a universal translation inhibitor. While the cytosine of amicetin mimics C75 of the 3′-CCA tail of the P-site tRNA akin to other nucleoside antibiotics, we advance a hypothesis that amicetin's unique interaction with the ribosomal protein uL16 exploits an untapped mechanism for selectively targeting the bacterial ribosome. A complex molecule comprised of a nucleoside, carbohydrates and amino acids, amicetin is also chemically unstable. Our initial attempts to stabilize and simplify this scaffold are presented with the ultimate goal of rebuilding the compound with improved penetrance to bacterial cells. If successful, this scaffold ...

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“…15 An unconstrained model of the 35-mer RNA-amicetin complex showed folding of the antibiotic within the complex. 15 We have now carried out a solution state NMR structure determination of amicetin to characterise its solution state properties, and the results obtained are described below.…”

Section: Introductionmentioning

confidence: 99%

NMR structure of the peptidyl transferase RNA inhibitor antibiotic amicetin

Shammas¹,

Donarski²,

Ramesh³

2006

Magnetic Reson in Chemistry

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In this communication, we report the solution state NMR structure determination of the peptidyl transferase RNA inhibitor antibiotic amicetin. We have successfully characterised the NMR spectrum of amicetin using a range of homo- and heteronuclear NMR techniques. Using experimental ROE-based distance and 1H--1H scalar coupling derived dihedral angle geometrical constraints as input into the three-dimensional structure determination protocol, we have generated an energy-minimised average structure of the antibiotic. Amicetin adopts a stable well-folded conformation in solution, mediated by a network of hydrogen bonds caused by proton donor and acceptor groups at either end of the molecule. The NMR structure of amicetin shows that the cytosine moiety occupies the critical turn position within the fold, which may be structurally significant for interaction with peptidyl transferase ribosomal RNA. The structure is distinctly different from the published X-ray crystal structure of amicetin in which it adopts a linear, extended chain-like conformation with a number of intermolecular hydrogen bonds. In addition to structure, we have probed the dynamics of amicetin in solution and have observed retarded exchange of the amide proton involved in folding. We have also characterised the ionisation properties of amicetin by carrying out NMR pH titration and measuring the pKa of the primary and tertiary amino groups, 7.27 and 7.52, respectively, which are in agreement with the reported values in literature. Solving the NMR structure of amicetin provides a valuable opportunity to determine the structure of its complex with RNA in solution state.

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NMR and Molecular Modelling Studies of the Binding of Amicetin Antibiotic to Conserved Secondary Structural Motifs of 23S Ribosomal RNAs

Cited by 14 publications

References 27 publications

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

Characterization of the Amicetin Biosynthesis Gene Cluster from Streptomyces vinaceusdrappus NRRL 2363 Implicates Two Alternative Strategies for Amide Bond Formation

Dissecting the Nucleoside Antibiotics as Universal Translation Inhibitors

NMR structure of the peptidyl transferase RNA inhibitor antibiotic amicetin

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