Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5′ S ,6′ S )-5′- C -glycyluridine (GlyU). GlyU is further modified with an N -alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N -alkylamine installation for ADR–GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S -adenosyl- l -methionine (AdoMet) as the direct precursor of the N -alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway. Rather than a typical S -adenosylmethionine-dependent alkyltransferase, the installation of the N -alkylamine linker in several nucleoside antibiotics is catalyzed via γ-replacement by a pyridoxal-5′-phosphate-dependent aminobutyryltransferase.