Project Summary/Abstract Peptidyl nucleosides (PNs) are naturally occurring antifungal agents active against multiple pathogenic fungi such as the causal agent of “Valley Fever”. They also exhibit potent synergistic effects with clinically approved antifungal drugs. Our long-term goal is to provide a comprehensive understanding of both the biosynthesis of PNs and their mode of action. The current application focuses on the biosynthesis of PNs. Understanding PN biosynthetic pathways will provide a basis for creating structurally diverse PN analogs through engineered biosynthesis, semi synthesis and genome mining. In the previous funding cycle, we found that PNs are biosynthesized through cryptic phosphorylation and carbohydrate tailoring by oxidative C-C bond cleavage. On the basis of these findings, in this application, we will perform functional and mechanistic characterization of the biosynthetic enzymes to provide the foundation for genome mining discovery of novel nucleoside natural products and chemoenzymatic synthesis of unnatural PNs. In Aim 1, the mechanism of removal of cryptic phosphorylation and the generality of cryptic phosphorylation in other nucleoside biosynthesis pathways will be investigated. In Aim 2, the radical mediated divergent biosynthesis of nucleoside natural products will be investigated by studying the mechanism of oxidative C-C bond cleavage and genome mining characterization of homologous enzymes. In Aim 3, the mechanism of amide ligation by NikS/PolG enzymes will be characterized, and their potentials for use in the chemoenzymatic preparation of PNs will be investigated. The proposed research is significant because it will provide a basis for the future biosynthetic and chemoenzymatic generation of novel therapeutic PNs as well as genome mining discovery of novel antifungal nucleoside natural products.