Abstract 2298 Complete in vitro reconstitution of the apramycin biosynthetic pathway demonstrates incorporation of a β-D-sugar nucleotide

Apramycin is a widely used pseudotrisaccharide aminoglycoside antibiotic composed of 2-deoxystreptamine, an octose, and D-4-amino-4-deoxyglucose. The two sugars are linked by an O-glycosidic bond between the C8' and C1" anomeric carbons. The biosynthetic pathway of the pseudodisaccharide aprosamine has been elucidated through years of effort by biochemists; however, how the 4-amino-4-deoxyglucose moiety is assembled has remained unclear. Herein, the unassigned apramycin biosynthetic proteins are characterized in vitro leading to a complete understanding of apramycin biosynthesis. The 4-amino-4-deoxyglucose moiety is derived from D-glucose 6-phosphate (G6P). Equilibration of G6P with β-D-glucose 1-phosphate (β-G1P) is catalyzed by AprJ, and AprK catalyzes a nucleotidylyl transfer reaction to produce NDP-β-D-glucose from β-G1P. Although AprK has relatively broad substrate tolerance for nucleotide donors, it tends to prefer pyrimidines over purines. AprD5 and AprL are identified as catalyzing the modification of NDP-β-D-glucose so as to introduce an amino group at the 4" position. Characterization of the glycoside hydrolase family 65 protein AprO demonstrated that NDP-β-D-4"-amino-4"-deoxyglucose serves as the sugar donor to produce apramycin 5-phosphate. When AprO was supplied with NDP-β-D-glucose instead of NDP-β-D-4"-amino-4"-deoxyglucose, saccharocin 5-phosphate was produced. While an NDP-α-D-hexose or an NDP-β-L-hexose is the typical sugar donor in biological glycosylation reactions, AprO surprisingly utilizes a β-D-sugar nucleotide to form the 8',1"-O-glycosidic linkage of apramycin. Therefore, NDP-hexose donors are not limited to α-D-sugar nucleotides in natural product biosynthesis. These findings may open the door to the chemoenzymatic preparation of sugar nucleotides that are otherwise not found in nature.