Biosynthesis of the Central Piperidine Nitrogen Heterocycle in Series a Thiopeptides

.Summary of main observation and conclusion Thiopeptides, arising from complex posttranslational modifications of a genetically encoded precursor peptide, are of great interest due to their structural complexity and important biological activities. All of these antibiotics share a macrocyclic peptidyl core that contains a central, six-membered nitrogen heterocycle and are classified into five series a-e based on the oxidation state of the central nitrogenous ring. Here, we report that the biosynthesis of the central piperidine heterocycle of series a thiopeptides relies on the activity of homologues of an F420H2-dependent reductase TppX(4) by exploiting and characterizing the piperidine-containing thiopeptin biosynthetic gene (tpp) cluster in Streptomyces tateyamensis. In vitro reconstruction of TppX(4) activity demonstrated that the piperidine heterocycle of thiopeptins was transformed from a dehydropiperidine heterocycle, and TppX(4) tolerated the changes in the C-termini and macrocyclic peptidyl core of substrate and also tolerated dehyropiperidine-containing monocyclic or bicyclic thiopeptides. The identification of TppX(4) and its substrate tolerance enriches the biosynthetic toolbox for development of additional thiopeptide analogs for clinical drug screening.