Studies of a siderophore-producing cyclization domain: A refined proposal of substrate binding

Abstract

Nonribosomal peptide synthetase (NRPS) heterocyclization (Cy) domains generate biologically important ox-/thiazoline modifications in natural product pathways, producing compounds that target disease and siderophores that are important for bacterial pathogenicity. The Cy domain shares the same fold as the NRPS condensation domain, but how the Cy domain catalyzes consecutive condensation and cyclodehydration reactions is unknown. To further understanding of Cy domain catalysis, we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. We find the high-resolution structure of Cy2 adopts a conformation that enables exploration of binding of the extended, thiazoline-containing cyclodehydration intermediate for catalysis and the acceptor carrier protein to which it is tethered. We also report the complementary electrostatic interfaces between Cy2 and its donor carrier protein to mediate donor binding. Lastly, we explore domain flexibility through the normal mode approximation and identify small-molecule fragment binding sites to inform antibiotic design targeting Cy function. Our results suggest how carrier protein binding may influence global conformation, with consequences for active site catalytic states and inhibitor development.