Thiopeptin, a New Feed-Additive Antibiotic: Biological Studies and Field Trials

Mine, Keisuke; Miyairi, Norimasa; Takano, Norio; Mori, Shinya; Watanabe, Naoaki

doi:10.1128/aac.1.6.496

Cited by 17 publications

(13 citation statements)

References 2 publications

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“…(7) 1. 35 d (7) CD3OD -CDCI3(I : 4) 5.02 dd (9,13) 3.69 dd (9,11) 3.21 dd (11, 13) 1.40d(7) through Thz (3) in thiostrepton and thiopeptins was complicated by the almost zero coupling between H-2 and H-3, making the CHNH proton doublets difficult to distinguish from the pair for the thiostreptine (Thstn) residue (see Table 6). In the case of thiostrepton A, (see companion …”

Section: Pipmentioning

confidence: 99%

Total structure of the highly modified peptide antibiotic components of thiopeptin.

Hensens¹,

Albers-Schönberg²

1983

J. Antibiot.

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On the basis of 'H and 13C NMR evidence, the structures of two series of the highly modified sulfur-containing peptide antibiotic thiopeptin were elucidated.The thiopeptins produced by Streptomyces tateyamensis belong to a group of highly modified sulfur containing peptide antibiotics'), the structures of several having only recently been determined by X-ray crystallography or physical methods=`'). They inhibit primarily Gram-positive bacteria'> and exhibit no significant differences in their inhibition of protein synthesis in cell-free Escherichia coli10). Thiopeptin B, the major component is valuable as a feed-additive because of its marked growth promoting action in swine and chickens11) and was recently shown to be effective as a lactic-acidosis preventive in sheep and cattle12).Thiopeptin B and four minor components A,, A, A> and A, were characterized by MIYAIRI et al.9.13) and shown primarily by acid hydrolysis experiments") to be closely related to the antibiotic thiostrepton, whose structure, with the exception of the side chain, was determined by X-ray crystallography by ANDERSON and coworkers-2). TORi et al.,3) from a "C NMR study of the antibiotic, proposed a sequence consisting of two dehydroalanine (Deala) residues for the side chain which was subsequently confirmed by our own 'H NMR study at 300 MHz (see preceding paper). Of the spectroscopic techniques available to us it soon became apparent that only 'H and 1>C NMR spectroscopy were to be potentially useful in the structural elucidation of the thiopeptins. The molecules were not sufficiently volatile to yield a molecular ion even under field-desorption conditions and only a fragment involving the dihydroquinoline moiety and adjacent peptide was identifiable (see below). Moreover, despite the fact that some of the a components were obtained in crystalline form, they were found not to be suitable for X-ray crystallography. A strategy, involving 'H NMR comparison with thiostrepton, was therefore adopted in order to delineate their structural differences. Pertinent details of confirmatory 1>C NMR evidence are also discussed") whereas a full discussion of the 11C NMR assignments is the subject of a companion paper. Results and DiscussionThiopeptin components were separated by silica gel chromatography similar to that previously described') and found to be homogeneous by TLC. 'H NMR analysis of the components from various batches however, indicated a two compound system in the majority of cases which was confirmed by HPLC. Consistent differences in the 'H NMR spectra allowed them to be grouped into two distinct series arbitrarily designated by the subscripts a and b and some of their physical and chemical properties are summarized in Table 1. In some batches all components from the a series and only Al,, from the b

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Section: Pipmentioning

confidence: 99%

Total structure of the highly modified peptide antibiotic components of thiopeptin.

Hensens¹,

Albers-Schönberg²

1983

J. Antibiot.

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“…Nevertheless, antibiotics often have alternative molecular targets and, like other secondary metabolites, elicit numerous "unexpected" effects on microbial differentiation (1)(2)(3)(4) and mammalian cell function (1). Here we describe how a single transcriptional activator can interact with diverse thiopeptide antibiotics to elicit autogenous expression of its own promoter as well as a modulon in Streptomyces lividans (SL).…”

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Broad Spectrum Thiopeptide Recognition Specificity of theStreptomyces lividans TipAL Protein and Its Role in Regulating Gene Expression

Chiu

Folcher

Katoh

et al. 1999

Journal of Biological Chemistry

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Microbial metabolites isolated in screening programs for their ability to activate transcription of the tipA promoter (ptipA) in Streptomyces lividans define a class of cyclic thiopeptide antibiotics having dehydroalanine side chains ("tails"). Here we show that such compounds of heterogeneous primary structure (representatives tested: thiostrepton, nosiheptide, berninamycin, promothiocin) are all recognized by TipAS and TipAL, two in-frame translation products of the tipA gene. The Nterminal helix-turn-helix DNA binding motif of TipAL is homologous to the MerR family of transcriptional activators, while the C terminus forms a novel ligand-binding domain. ptipA inducers formed irreversible complexes in vitro and in vivo (presumably covalent) with TipAS by reacting with the second of the two C-terminal cysteine residues. Promothiocin and thiostrepton derivatives in which the dehydroalanine side chains were removed lost the ability to modify TipAS. They were able to induce expression of ptipA as well as the tipA gene, although with reduced activity. Thus, TipA required the thiopeptide ring structure for recognition, while the tail served either as a dispensable part of the recognition domain and/or locked thiopeptides onto TipA proteins, thus leading to an irreversible transcriptional activation. Construction and analysis of a disruption mutant showed that tipA was autogenously regulated and conferred thiopeptide resistance. Thiostrepton induced the synthesis of other proteins, some of which did not require tipA.Directed searches for microbial secondary metabolites that inhibit bacterial growth led to the discovery of antibiotics and thus gave rise to the traditional interpretation that their only biological relevance is to inhibit growth of competing organisms. Nevertheless, antibiotics often have alternative molecular targets and, like other secondary metabolites, elicit numerous "unexpected" effects on microbial differentiation (1-4) and mammalian cell function (1). Here we describe how a single transcriptional activator can interact with diverse thiopeptide antibiotics to elicit autogenous expression of its own promoter as well as a modulon in Streptomyces lividans (SL). 1Thiopeptides are a family of antibiotics composed of a ring structure containing highly modified amino acids and a linear peptide containing dehydroalanines extending from the ring at a pyridyl group ("tail") ( Fig. 1). They were first discovered as antibiotics synthesized by diverse bacteria including Streptomyces, Bacillus, and Micrococcus. These compounds later proved to be effective growth promotants for domestic animals (2-4), an effect whose biological basis is not clear. Thiostrepton, whose antibiotic activity is best understood, acts by binding tightly to the procaryotic ribosome and thus inhibiting translation (5-8). In a thiostrepton-producing organism, Streptomyces azureus, methylation of a specific nucleotide in the 23 S rRNA can provide resistance. Such methylated ribosomes do not bind and are therefore not sensitive to thiostrep...

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“…These compounds are employed as antibiotics, growth promotants, and prophylactic agents for livestock (Mine et al, 1972;Muir et al, 1980). Thiostrepton is well-known for its ability to bind to prokaryotic ribosomes, thereby inhibiting protein synthesis, and has been used widely as a tool to study ribosome function (Cundliffe, 1987).…”

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Characterization of the Covalent Binding of Thiostrepton to a Thiostrepton-Induced Protein from Streptomyces lividans

et al. 1996

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Thiostrepton is a highly modified multicyclic peptide antibiotic synthesized by diverse bacteria. Although best known as an inhibitor of protein synthesis, thiostrepton is also a potent activator of gene expression in Streptomyces lividans. In these studies, we characterize the nature of the interaction between thiostrepton and two proteins that it induces, TipAL and TipAS. In the absence of added cofactors, thiostrepton formed a complex with either TipAL or TipAS in aqueous solution. The TipA-thiostrepton complex was not dissociated by denaturants such as SDS, urea, or disulfide reducing agents. The mass of the TipAS-thiostrepton complex as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS) was equivalent to the sum of TipAS and thiostrepton. Thiostrepton also reacted spontaneously with free cysteine (but not with other amino acids tested) to generate stable compounds having masses equivalent to thiostrepton plus 3 to 4 cysteines. Blocking experiments indicated that complex formation required dehydroalanine residues on thiostrepton and cysteine residues on TipAS. When the TipAS-thiostrepton complex was digested with trypsin and analyzed by MS, the thiostrepton adduct was found bound only to the unique cysteine-containing TipAS peptide fragment. Amino acid analysis confirmed that the TipAS-thiostrepton complex contained lanthionine, the product of a reaction between dehydroalanine and cysteine. Together, these data document a covalent attachment of thiostrepton to TipA proteins mediated by bond formation between dehydroalanine of thiostrepton and cysteine of TipAS. Implications regarding the function of TipAS as a thiostrepton (electrophile)-sequestering protein and thiostrepton-mediated activation of TipAL as a model of irreversible transcriptional activation are discussed.

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Thiopeptin, a New Feed-Additive Antibiotic: Biological Studies and Field Trials

Cited by 17 publications

References 2 publications

Total structure of the highly modified peptide antibiotic components of thiopeptin.

Total structure of the highly modified peptide antibiotic components of thiopeptin.

Broad Spectrum Thiopeptide Recognition Specificity of theStreptomyces lividans TipAL Protein and Its Role in Regulating Gene Expression

Characterization of the Covalent Binding of Thiostrepton to a Thiostrepton-Induced Protein from Streptomyces lividans

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