Biosynthesis of ureido containing natural products: Mechanistic studies and structural studies - Natural products (aka specialized metabolites) are small molecules produced by bacteria, fungi, plants, and animals that have found many uses in agricultural, veterinary, and human health. More than half of all currently FDA approved drugs are natural products or derived from natural products. In addition, to their far- ranging biological activities (anti-cancer to anti-bacterial to anti-viral) the pathways that organisms utilize to construct natural products from simple, readily available building blocks, particularly those used by microbes, have triggered much interest. Both as a way to understand the mechanisms and enzymes to allow manipulation of the proteins and pathways to produce new-to-nature natural products with altered biological activity and from an intellectual standpoint. In this proposal, we dissect the mechanism of ureido bond formation in the biosynthesis of three families of bioactive bacterial natural products, the anabaenopeptins, the syringolins, and the muramycins, which derive from diverse bacterial genera. These biologically active molecules contain the proteolytically stable ureido bond, which is a key contributor to their biological activity. While the mechanism of ureido formation in biotin biosynthesis has been studied, we hypothesize that the biosynthesis of the anabaenopeptins, syringolins, and muramycins, which are all assembled through the action of a non-ribosomal peptide synthetase (NRPS), are distinct due to the fact that the intermediates are tethered to the NRPS in contrast to the untethered biotin intermediate, 7,8-diaminononanoate. Our preliminary data suggests that the mechanism utilized in the biosynthesis of the anabaenopeptins is distinct from the mechanism previously proposed for the biosynthesis of the syringolins. This proposal describes a multidisciplinary approach consisting of protein overexpression, isotope labeling, and chemical trapping in Aim 1, which is complemented by cryo-EM, and site directed mutagenesis in Aim 2. These specific aims will allow the elucidation of the mechanism utilized during ureido bond formation while identifying the protein residues and motifs involved in substrate binding and catalysis in proteins found in different bacterial genera and with different protein architectures. The identification of the motifs will allow the proper annotation of ureido bond forming condensation domains found in deposited sequenced genomes, which is an issue as they are currently annotated as having amide bond forming activity by popular annotation software like AntiSMASH. This prevents the accurate prediction of compound structure from genomic data and represents a current hurdle in the field of natural products. The knowledge gained in this proposal will set the stage for the future engineering of new natural products with altered biological activity by introducing ureido bonds to increase proteolytic stability and alter physiochemical properties.
