Kanosamine (3-amino-3-deoxy-D-glucose) is a characteristic sugar unit found in kanamycins, a group of aminoglycoside antibiotics. The kanosamine moiety originates from D-glucose in kanamycin biosynthesis. However, the timing of the replacement of the 3-OH group of the D-glucose-derived biosynthetic intermediate with the amino group is elusive. Comparison of biosynthetic gene clusters for related aminoglycoside antibiotics suggests that the nicotinamide adenine dinucleotide (NAD + )dependent dehydrogenase KanD2 and the pyridoxal 5′-phosphate (PLP)-dependent aminotransferase KanS2 are responsible for the introduction of the amino group at the C3 position of kanosamine. In this study, we demonstrated that KanD2 and KanS2 convert kanamycin A, B, and C to the corresponding 3″-deamino-3″-hydroxykanamycins (3″-hks) in the presence of PLP, 2-oxoglutarate, and NADH via a reverse reaction in the pathway. Furthermore, we observed that all of the 3″-hks are oxidized by KanD2 with NAD + , but D-glucose, UDP-D-glucose, D-glucose 6-phosphate, and D-glucose 1-phosphate are not. Crystal structure analysis of KanD2 complexed with 3″-hkB and NADH illustrated the selective recognition of pseudotrisaccharides, especially the D-glucose moiety with 2-deoxystreptamine, by a combination of hydrogen bonds and CH−π interactions. Overall, it was clarified that the kanosamine moiety of kanamycins is constructed after the glucosylation of the pseudodisaccharide biosynthetic intermediates in kanamycin biosynthesis.

Kanamycin A is the major 2‐deoxystreptamine (2DOS)‐containing aminoglycoside antibiotic produced by Streptomyces kanamyceticus. The 2DOS moiety is linked with 6‐amino‐6‐deoxy‐d‐glucose (6ADG) at O‐4 and 3‐amino‐3‐deoxy‐d‐glucose at O‐6. Because the 6ADG moiety is derived from d‐glucosamine (GlcN), deamination at C‐2 and introduction of C‐6‐NH2 are required in the biosynthesis. A dehydrogenase, KanQ, and an aminotransferase, KanB, are presumed to be responsible for the introduction of C‐6‐NH2, although the substrates have not been identified. Here, we examined the substrate specificity of KanQ to better understand the biosynthetic pathway. It was found that KanQ oxidized kanamycin C more efficiently than the 3′′‐deamino derivative. Furthermore, the substrate specificity of an oxygenase, KanJ, that is responsible for deamination at C‐2 of the GlcN moiety was examined, and the crystal structure of KanJ was determined. It was found that C‐6‐NH2 is important for substrate recognition by KanJ. Thus, the modification of the GlcN moiety occurs after pseudo‐trisaccharide formation, followed by the introduction of C‐6‐NH2 by KanQ/KanB and deamination at C‐2 by KanJ.