Margherita Sosio1, Eleonora Gaspari2, Marianna Iorio2, Silvia Pessina2, Marnix H Medema3, Alice Bernasconi2, Matteo Simone2, Sonia I Maffioli4, Richard H Ebright5, Stefano Donadio4. 1. Naicons Srl, Viale Ortles 22/4, 20139 Milan, Italy; KtedoGen Srl, Viale Ortles 22/4, 20139 Milan, Italy. Electronic address: msosio@naicons.com. 2. Naicons Srl, Viale Ortles 22/4, 20139 Milan, Italy. 3. Bioinformatics Group, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands. 4. Naicons Srl, Viale Ortles 22/4, 20139 Milan, Italy; KtedoGen Srl, Viale Ortles 22/4, 20139 Milan, Italy. 5. Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
Abstract
Pseudouridimycin (PUM) is a selective nucleoside-analog inhibitor of bacterial RNA polymerase with activity against Gram-positive and Gram-negative bacteria. PUM, produced by Streptomyces sp. ID38640, consists of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 5'-aminopseudouridine. We report the characterization of the PUM gene cluster. Bioinformatic analysis and mutational knockouts of pum genes with analysis of accumulated intermediates, define the PUM biosynthetic pathway. The work provides the first biosynthetic pathway of a C-nucleoside antibiotic and reveals three unexpected features: production of free pseudouridine by the dedicated pseudouridine synthase, PumJ; nucleoside activation by specialized oxidoreductases and aminotransferases; and peptide-bond formation by amide ligases. A central role in the PUM biosynthetic pathway is played by the PumJ, which represents a divergent branch within the TruD family of pseudouridine synthases. PumJ-like sequences are associated with diverse gene clusters likely to govern the biosynthesis of different classes of C-nucleoside antibiotics.
Pseudouridimycin (n class="Chemical">PUM) is a selective nucleoside-analog inhibitor of bacterial RNA polymerase with activity against Gram-positive and Gram-negative bacteria. PUM, produced by Streptomyces sp. ID38640, consists of a formamidinylated, N-hydroxylated Gly-Glndipeptide conjugated to 5'-aminopseudouridine. We report the characterization of the PUM gene cluster. Bioinformatic analysis and mutational knockouts of pum genes with analysis of accumulated intermediates, define the PUM biosynthetic pathway. The work provides the first biosynthetic pathway of a C-nucleoside antibiotic and reveals three unexpected features: production of free pseudouridine by the dedicated pseudouridine synthase, PumJ; nucleoside activation by specialized oxidoreductases and aminotransferases; and peptide-bond formation by amide ligases. A central role in the PUM biosynthetic pathway is played by the PumJ, which represents a divergent branch within the TruD family of pseudouridine synthases. PumJ-like sequences are associated with diverse gene clusters likely to govern the biosynthesis of different classes of C-nucleoside antibiotics.
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