C3′‐Deoxygenation of Paromamine Catalyzed by a Radical S‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

Abstract

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.

Following the electrons: peculiarities in the catalytic cycles of radical SAM enzymes

“…Furthermore, isotope tracer experiments have demonstrated that the C4 hydron of paromamine as opposed to a solvent hydron is incorporated into the 5′-deoxyadenosine produced during turnover. 9,10 This result essentially rules out the possibility that the net reduction of SAM is entirely due to uncoupled reactions competing with the dehydration reaction. It is also worth noting that AprD4 is not the first radical SAM enzyme to differ from its B 12 -dependent counterpart in this peculiar way.…”

“…1 & 4). 9–11 Therefore, the AprD4 reaction is very similar to that of the B 12 -dependent diol-dehydratases 54 with the important difference that unlike adenosyl-cobalamin, SAM is not regenerated with each turnover. Furthermore, isotope tracer experiments have demonstrated that the C4 hydron of paromamine as opposed to a solvent hydron is incorporated into the 5′-deoxyadenosine produced during turnover.…”

“…AprD4 together with AprD3 is responsible for the reduction of paromamine to lividamine during the biosynthesis of apramycin in species of Streptomyces . 9–11,53 In doing so, AprD4 serves as a redox neutral dehydratase converting paromamine to a keto intermediate prior to its reduction by the NADPH-dependent reductase AprD3 (see Figs. 1 & 4).…”

“…Reactions catalyzed by radical SAM enzymes involve either a redox neutral transformation of the nominal substrate or its oxidation. For example, BtrN 8 catalyzes a dehydrogenation whereas AprD4 9–11 catalyzes a dehydration. Reduction of SAM to L-methionine (Met) and 5′-deoxydenosine (5′-dAdo) is a hallmark of the associated catalytic cycles and is depicted here as a stepwise process involving a 5′-deoxyadenosyl radical intermediate (5′-dAdo-CH 2 • ).…”

“…1). Likewise, redox neutral transformations such as the dehydration reaction catalyzed by AprD4 9–11 (see Fig. 1) in principle should be able to utilize SAM catalytically; however, there are relatively few examples where this has been shown to be the case.…”