Conserved Mechanism of 2′-Phosphorylation-Aided Amide Ligation in Peptidyl Nucleoside Biosynthesis

Abstract

Peptidyl nucleoside antifungals, represented by nikkomycins and polyoxins, consist of an unusual six-carbon nucleoside [aminohexuronic acid (AHA)] ligated to a nonproteinogenic amino acid via an amide bond. A recent study suggested that AHA is biosynthesized through cryptic phosphorylation, where a 2′-phosphate is introduced early in the pathway and required to form AHA. However, whether 2′-phosphorylation is necessary for the last step of biosynthesis, the formation of the amide bond between AHA and nonproteinogenic amino acids, remains ambiguous. Here, we address this question with comprehensive in vitro and in vivo characterizations of PolG and NikS, which together provide strong evidence that amide ligation proceeds with 2′phosphorylated substrates in both pathways. Our results suggest that 2′-phosphorylation is retained for the entirety of both nikkomycin and polyoxin biosynthesis, providing important insights into how cryptic phosphorylation assists with nucleoside natural product biosynthesis. P eptidyl nucleosides (PNs), such as nikkomycins and polyoxins, exhibit selective antifungal activities with no known side effects to higher eukaryotes. Consequently, polyoxin D [1 (Figure 1A)] is a safely used fungicide in Communication pubs.acs.org/biochemistry

Cryptic Phosphorylation-Mediated Divergent Biosynthesis of High-Carbon Sugar Nucleoside Antifungals

“…In the nikkomycin and polyoxin pathways, AHA had long been thought of as a biosynthetic intermediate due to its accumulation in the culture medium of mutant strains that abolish the biosynthesis of the N-terminal amino acid moiety. However, the recent discovery of the cryptic phosphorylation 9 and subsequent kinetic characterization of the amide ligases 16 revealed that AHA is actually a shunt metabolite. Although a phosphatase has not been identified for either of these pathways, dephosphorylation most likely occurs at the end of the pathways.…”

Biosynthesis and Genome Mining Potentials of Nucleoside Natural Products

“…Similar phosphorylation of biosynthetic intermediates was also found in some of the LipL‐containing pathways, such as muraymycin [43] and caprazamycin [34] biosynthesis, and has been proposed as a self‐resistance mechanism against the toxic effects of the biosynthetic intermediates. Although the role of 2'‐phosphorylation in the nikkomycin pathway is unclear, it is essential for the activities of the downstream enzymes and conserved in nikkomycin, polyoxin, and malayamycin pathways [24a,35a] . Since kinases with unknown functions are frequently found in many other nucleoside BGCs, [35b] similar cryptic phosphorylation is likely operating in other nucleoside pathways.…”

Biosyntheses of azetidine-containing natural products

“…45 Nonetheless, the 2′-phosphorylation containing nucleoside skeleton ( 106 ) was subsequently affirmed as the physiological substrate of PolG and the cryptic phosphorylation is retained till the final step of polyoxin biosynthesis. 46 But the characterization of PolG is ambiguous whether this enzyme also assembles the nucleoside skeleton and POIA or not. In addition, the oxygenations of the 5-methyl group in the nucleoside skeleton to hydroxyl or carboxyl as found in polyoxins A, F and I are obscure, probably by a promiscuous oxygenase located outside of the pol cluster.…”