C3′-deoxygenation of aminoglycosides results in their decreased susceptibility to phosphorylation thereby increasing their efficacy as antibiotics. However, the biosynthetic mechanism of C3′-deoxygenation is unknown. To address this question, aprD4 and aprD3 genes from the apramycin gene cluster in Streptomyces tenebrarius were expressed in E. coli and the resulting gene products were characterized in vitro. AprD4 is shown to be a radical SAM enzyme catalyzing homolysis of SAM to 5′-deoxyadenosine (5′-dAdo) in the presence of paromamine. [4′-2H]-Paromamine was prepared and used to demonstrate that its C4′-H is transferred to 5′-dAdo by AprD4, during which the substrate is dehydrated to a product consistent with 4′-oxolividamine. In contrast, paromamine is reduced to a deoxy product when incubated with AprD4/AprD3/ NADPH. These results indicate that AprD4 is the first radical SAM diol-dehydratase and along with AprD3 is responsible for 3′-deoxygenation in the biosynthesis of aminoglycosides.

AprD4 is a radical S-adenosyl-L-methionine (SAM) enzyme catalyzing C3′-deoxygenation of paromamine to form 4′oxo-lividamine. It is the only 1,2-diol dehydratase in the radical SAM enzyme superfamily that has been identified and characterized in vitro. The AprD4 catalyzed 1,2-diol dehydration is a key step in the biosynthesis of several C3′-deoxy-aminoglycosides. While the regiochemistry of the hydrogen atom abstraction catalyzed by AprD4 has been established, the mechanism of the subsequent chemical transformation remains not fully understood. To investigate the mechanism, several substrate analogues were synthesized and their fates upon incubation with AprD4 were analyzed. The results support a mechanism involving formation of a ketyl radical intermediate followed by direct elimination of the C3′-hydroxyl group rather than that of a gem-diol intermediate generated via 1,2migration of the C3′-hydroxyl group to C4′. The stereochemistry of hydrogen atom incorporation after radical-mediated dehydration was also established.