Biochemical and Structural Analysis of a Dehydrogenase, KanD2, and an Aminotransferase, KanS2, That Are Responsible for the Construction of the Kanosamine Moiety in Kanamycin Biosynthesis

Abstract

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.

A Machine Learning Bioinformatics Method to Predict Biological Activity from Biosynthetic Gene Clusters

“…The eighth largest cluster is associated with antibacterial activity, and the sub-PFAM domains in this cluster all transfer amines to sugars in aminoglycoside antibiotics, e.g., kanosamine, part of the antibiotic kanamycin ( Figure 4 B). 35 Therefore clusters 6 and 8 both produce substructures of a single class of natural product. In the case of the polyene macrolides, the mycosamine sugar has been shown to be essential for activity, likely through its interactions with sterols in the fungal membrane.…”

“…BGCs containing this mycosamine-producing sub-PFAM domain are all polyene macrolides with similar structures (Figure S5). The eighth largest cluster is associated with antibacterial activity, and the sub-PFAM domains in this cluster all transfer amines to sugars in aminoglycoside antibiotics, e.g., kanosamine, part of the antibiotic kanamycin (Figure B) . Therefore clusters 6 and 8 both produce substructures of a single class of natural product.…”

Biosynthesis of cyclitols

“…23). The sugar moieties are further modied to produce matured kanamycins [105][106][107] and gentamicins. 103,[108][109][110][111][112][113][114][115][116][117] 3.4.3 Biosynthesis of hygromycin B.…”

“…23). The sugar moieties are further modified to produce matured kanamycins 105–107 and gentamicins. 103,108–117…”

Stepwise Post‐glycosylation Modification of Sugar Moieties in Kanamycin Biosynthesis

“…This intermediate is then aminated by pyridoxal 5'-phosphate (PLP)-dependent aminotransferase KanS2 (UniProt: Q6L733) to yield kanamycin C (6; Scheme 1). [9] KanD2 and KanS2 also recognize kanamycin A (8), kanamycin B (7), and kanamycin C (6), and convert those to the 3''-deamino-3''-hydroxy-derivatives in the presence of NADH and α-ketoglutarate (α-KG). Because KanD2 does not oxidize Glc, G6P, or UDP-Glc, the modification event to construct the kanosamine moiety occurs after glycosylation.…”