Caprazamycin is a nucleoside antibiotic that inhibits phospho-N-acetylmuramyl-pentapeptide translocase (MraY). The biosynthesis of nucleoside antibiotics has been studied however remains to be removed from completion. The current examine characterised enzymes Cpz10, Cpz15, Cpz27, Mur17, Mur23 out of caprazamycin/muraymycin biosynthetic gene cluster, notably the nonheme αKG-dependent enzyme Cpz10. Cpz15 is a β-hydroxylase changing uridine mono-phosphate to uridine 5′ aldehyde, then incorporating with threonine by Mur17 (Cpz14) to kind 5′-C-glycyluridine. Cpz10 hydroxylates artificial 11 to 12 in vitro. Main product 13 derived from mutant Δcpz10 is phosphorylated by Cpz27. β-Hydroxylation of 11 by Cpz10 permits the maturation of caprazamycin, however decarboxylation of 11 by Mur23 oriented to muraymycin formation. Cpz10 recruits two iron atoms to activate dioxygen with regio-/stereo-specificity and commit electron/cost switch, respectively. The chemo-physical interrogations ought to tremendously advance our understanding of caprazamycin biosynthesis, which is conducive to pathway/protein engineering for creating simpler nucleoside antibiotics.