Biosynthesis of ureido containing natural products: Mechanistic studies and structural studies

This proposal aims to elucidate the molecular mechanisms of ureido bond formation within multiple non-ribosomal peptide synthetases (NRPSs). Aim 1 will focus on biochemical elucidation of the molecular mechanism of ureido bond formation within NRPSs from both Gram-positive and Gram-negative bacteria. NRPSs with different domain architectures will be explored biochemically in this aim. Aim 2 will identify key residues and motifs for ureido bond formation by a condensation domain using site-directed mutagenesis and single particle cryogenic electron microscopy (cryo-EM). Within Aim 2, structural characterization will investigate differences in domain architecture between different NRPSs and probe how these differences affect the molecular mechanism of ureido bond formation between bacterial species. High-resolution structures of the active sites of condensation domains will allow identification of key residues involved in ureido bond formation.