Cryptic Phosphorylation in Nucleoside Natural Product Biosynthesis

Abstract

Peptidyl nucleoside (PN) natural products, represented by the nikkomycins and polyoxins, are an important class of medicinally (nikkomycin Z) and agriculturally (polyoxin D) useful compounds with selective and potent antifungal activities. PNs are characterized by a highly conserved, six-membered nucleoside, amino hexuronic acid (AHA), coupled to a non-proteinogenic amino acid. AHA's biosynthesis has been a long-standing mystery in the field of nucleoside biosynthesis and understanding this pathway may provide critical insights into biosynthetically inspired preparations of novel antifungal therapeutics. I here present AHA's biosynthetic pathway in its entirety, which unexpectedly proceeds via a crytpic and unprecedented phosphorylation at the 2'-OH. These studies were supplemented with kinetic characterization of the amide ligases from the nikkomycin and polyoxin pathways, NikS and PolG, demonstrating a greater than 90-fold preference for 2'-phosphorylated nucleoside substrates and supporting the relevance of 2'-phosphorylation as a recognition tag in PN biosynthesis. I extended the relevance of cryptic phosphorylation to the nucleoside natural product malayamycin A, revealing that this pathway also proceeds via cryptic phosphorylation. My studies demonstrate that nikkomycins/polyoxins and malayamycin A share a divergent biosynthetic strategy that suggests novel and chemically interesting C-C bond cleaving oxidative reactions. I also present preliminary mechanistic characterizations of the oxygenases MalM and PolD/MalI that are responsible for these key oxidative transformations in AHA and malayamycin A biosynthesis. This work establishes cryptic phosphorylation as a novel paradigm for nucleoside natural product biosynthesis, and genome mining suggests that cryptic phosphorylation may be a biosynthetic strategy for nucleoside natural products in general.