Oxa-bridged macrolides are natural products produced by Actinobacteria, with biosynthesis involving large gene clusters and alpha-ketoglutarate-dependent non-heme iron (aKG-NHF) dioxygenases [1,2]. These enzymes catalyse the installation of the diether bridge, a process crucial for understanding biosynthesis and potentially enabling chemoenzymatic total synthesis or diether bridge installation in other molecules.
The nargenicin family of oxa-bridged macrolides comprises narrow-spectrum antibiotics effective against various Gram-positive pathogens. Nargenicin A1, the most well-studied member, features a unique ether-bridged cis-decalin moiety. This family includes analogues like streptoseomycin and branimycin, each with a ten-membered macrolactone ring, except branamycin, which has a nine-membered ring. The exact position of the ether bridge varies among the family; nargenicin and branimycin have an 8,13 linkage, while streptoseomycin bridge between positions 9 and 13 [1]. Despite challenges in chemical synthesis, the diether bridge is crucial for their antibacterial activity, possibly altering the molecular configuration to reduce affinity for the antibacterial target, replicative DNA polymerase.
In this study, we used X-ray crystallography to elucidate the structures of three αKG-NHF enzymes (NarN, BraN, and StmO3) responsible for incorporating the diether bridge into nargenicin, branamycin, and streptoseomycin, respectively. Mutations in NarN residues were found to abolish dioxygenase activity. The insights derived from these structures contributes to a detailed understanding of nargenicin macrolide biosynthesis and paves the way for biocatalytic optimization of a chemically difficult bond.