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Thethiopeptides are a family of ribosomally synthesized and post-translationallymodified peptide metabolites, and the vast majority of thiopeptidescharacterized to date possess one highly modified macrocycle. A fewmembers, including thiostrepton A, harbor a second macrocycle thatincorporates a quinaldic acid moiety and the four N-terminal residuesof the peptide. The antibacterial properties of thiostrepton A arewell established, and its recently discovered ability to inhibit theproteasome has additional implications for the development of antimalarialand anticancer therapeutics. We have conducted the saturation mutagenesisof Ala2 in the precursor peptide, TsrA, to examine which variantscan be transformed into a mature thiostrepton analogue. Although thethiostrepton biosynthetic system is somewhat restrictive toward substitutionsat the second residue, eight thiostrepton Ala2 analogues were isolated.The TsrA Ala2Ile and Ala2Val variants were largely channeled throughan alternate processing pathway wherein the first residue of the corepeptide, Ile1, is removed, and the resulting thiostrepton analoguesbear quinaldic acid macrocycles abridged by one residue. This is thefirst report revealing that quinaldic acid loop size is amenable toalteration during the course of thiostrepton biosynthesis. Both theantibacterial and proteasome inhibitory properties of the thiostreptonAla2 analogues were examined. While the identity of the residue atthe second position of the core peptide influences thiostrepton biosynthesis,our report suggests it may not be crucial for antibacterial and proteasomeinhibitory properties of the full-length variants. In contrast, thecontracted quinaldic acid loop can, to differing degrees, affect bothtypes of biological activity.
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