Analysis of the Pseudouridimycin Biosynthetic Pathway Provides Insights into the Formation of C-nucleoside Antibiotics

Margherita Sosio, Eleonora Gaspari, Marianna Iorio, Silvia Pessina, Marnix H. Medema, Alice Bernasconi, Matteo Simone, Sonia I. Maffioli, Richard H. Ebright, Stefano Donadio

Index: 10.1016/j.chembiol.2018.02.008

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Abstract

Pseudouridimycin (PUM) is a selective nucleoside-analog inhibitor of bacterial RNA polymerase with activity against Gram-positive and Gram-negative bacteria. PUM, produced byStreptomycessp. 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 ofpumgenes with analysis of accumulated intermediates, define the PUM biosynthetic pathway. The work provides the first biosynthetic pathway of aC-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 ofC-nucleoside antibiotics.