Biosynthesis of the Thiofuranose Core in Albomycin Requires a Versatile Enzyme AbmG That Catalyzes Net Dehydration via Cryptic Phosphorylation

Albomycins are unusual sulfur-containing nucleosides from the species of <i>Streptomyces</i> that exhibit potent antibiotic activities against both Gram-negative and Gram-positive bacteria including clinical pathogens. Previous studies demonstrated that the twitch radical SAM enzyme AbmM catalyzes an oxidative sulfur-for-oxygen swapping reaction converting CDP to a 4'-hydroxy-4'-thiocytidine 5'-diphosphate intermediate in the initial step of albomycin biosynthesis. However, the fate of this intermediate in the biosynthetic pathway has remained elusive. Herein, the above intermediate after 5'-dephosphorylation is shown to undergo AbmG-catalyzed transformations via a cryptic double phosphorylation of its 4'-hydroxyl group followed by C-O bond cleavage to yield 5'-oxo-4'-thiocytidine. X-ray crystal structure analysis and site-directed mutagenesis of AbmG revealed Glu188 as the general base to perform C5' deprotonation. Subsequent mechanistic studies using deuterated substrates demonstrated that the deprotonation at C5' is <i>pro</i>-<i>R</i> specific and likely occurs concerted with elimination of pyrophosphate from C4'. This study not only highlights a unique nucleoside kinase with lyase activity to complete an overall dehydration reaction but also fills the gaps in the biosynthesis of the atypical thiofuranose core essential to the biological activities of albomycins.