Complete Reconstitution of the Apramycin Biosynthetic Pathway Demonstrates the Unusual Incorporation of a β-d-Sugar Nucleotide in the Final Glycosylation Step

Apramycin is a widely used aminoglycoside antibiotic with applications in veterinary medicine. It is composed of a 4-amino-4-deoxy-d-glucose moiety and the pseudodisaccharide aprosamine, which is an adduct of 2-deoxystreptamine and an unusual eight-carbon bicyclic dialdose. Despite its extensive study and relevance to medical practice, the biosynthetic pathway of this complex aminoglycoside nevertheless remains incomplete. Herein, the remaining unknown steps of apramycin biosynthesis are reconstituted , thereby leading to a comprehensive picture of its biological assembly. In particular, phosphomutase AprJ and nucleotide transferase AprK are found to catalyze the conversion of glucose 6-phosphate to NDP-β-d-glucose as a critical biosynthetic intermediate. Moreover, the dehydrogenase AprD5 and transaminase AprL are identified as modifying this intermediate via introduction of an amino group at the 4″ position without requiring prior 6″-deoxygenation as is typically encountered in aminosugar biosynthesis. Finally, the glycoside hydrolase family 65 protein AprO is shown to utilize NDP-β-d-glucose or NDP-4"-amino-4"-deoxy-β-d-glucose to form the 8',1″--glycosidic linkage of saccharocin or apramycin, respectively. As the activated sugar nucleotides in all known natural glycosylation reactions involve either NDP-α-d-hexoses or NDP-β-l-hexoses, the reported chemistry expands the scope of known biological glycosylation reactions to NDP-β-d-hexoses, with important implications for the understanding and repurposing of aminoglycoside biosynthesis.