thiostrepton and dehydroalanine

Topics: Alanine; Anti-Bacterial Agents; Boronic Acids; Catalysis; Copper; Enterococcus faecalis; Microbial Sensitivity Tests; Nisin; Protein Processing, Post-Translational; Ribosomes; Solubility; Staphylococcus aureus; Thiazoles; Thiostrepton

Thiostrepton is a potent antibiotic against a broad range of Gram-positive bacteria, but its medical applications have been limited by its poor aqueous solubility. In this work, the first C(sp

Topics: Alanine; Anti-Bacterial Agents; Catalysis; Cobalt; Humans; Thiostrepton

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) are remarkably versatile non-canonical amino acids often found in antimicrobial peptides. This work presents the selective modification of Dha and Dhb in antimicrobial peptides through photocatalytic activation of organoborates under the influence of visible light. Ir(dF(CF

Topics: Alanine; Aminobutyrates; Borates; Catalysis; Coordination Complexes; Iridium; Light; Nisin; Oxidation-Reduction; Photochemical Processes; Ruthenium; Thiostrepton

Syntheses of dehydroalanine derivatives via a solid-support route, starting from selenocystein, and via conventional solution phase chemistry are described along with initial biological testing. The target compounds were designed as mimetics of the dehydroalanine side chain of the macrocyclic antibiotic thiostrepton that acts on the bacterial ribosome.

Topics: Alanine; Chromatography, Liquid; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Mimicry; Thiostrepton

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.

Topics: Alanine; Amino Acid Sequence; Amino Acids; Anti-Bacterial Agents; Bacterial Proteins; Chromatography, Thin Layer; Cysteine; Electrophoresis, Polyacrylamide Gel; Mass Spectrometry; Molecular Sequence Data; Peptide Mapping; Protein Binding; Streptomyces; Thiostrepton; Trans-Activators