Substrate specificity of radical S-adenosyl-l-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics

Abstract

A radical S-adenosyl-L-methionine dehydratase AprD4 and an NADPH-dependent reductase AprD3 are responsible for the C3′-deoxygenation of pseudodisaccharide paromamine in the biosynthesis of apramycin. These enzymes are involved in the construction of the characteristic structural motif that is not modified by 3′-phosphotransferase in aminoglycoside-resistant bacterial strains. AprD4 catalyzes the C3′-dehydration of paromamine via a radical-mediated reaction mechanism to give 4′-oxolividamine, which is then reduced by AprD3 with NADPH to afford lividamine. In the present study, the substrate specificity of this unique combination of enzymes has been investigated. AprD4 was found to recognize paromamine, neamine, kanamycin C, and kanamycin B to afford 5′-deoxyadenosine as one of products during the C3′-dehydration of aminoglycosides, but not 2′-N-acetylparomamine and paromomycin. Only paromamine and kanamycin C were converted to the corresponding C3′-deoxygenated compounds by AprD4 and AprD3. AprD3 recognizes the 4′-oxolividamine moiety, including the pseudotrisaccharide kanamycin C, and seems to reject the amino group at C6′ of neamine and kanamycin B. Chirally deuterium-labeled NADPH was used to identify that that AprD3 transfers the pro-S hydrogen atom of NADPH when reducing 4′-oxolividamine to give lividamine.

Parallel pathways in the biosynthesis of aminoglycoside antibiotics

“…Remarkably, AprQ is also able to act on the tripseudodisaccharide substrate gentamicin X2 and G418 and, in the latter case, produce a novel gentamicin analogue with a C6' carboxylate 20 . A similar observation was also made on AprD4 and AprD3, which are able to act on tripseudodisaccharide substrates, such as kanamycin C and kanamycin B 40 . The remarkable substrate promiscuity of apramycin biosynthetic enzymes demonstrates the great potential to produce novel AGs by pathway engineering efforts.…”

“…In contrast to the previous proposal in which the C3 deoxygenation occurs as the penultimate step, biochemical studies have shown that the C3 deoxygenation is catalyzed by the radical SAM diol dehydratase AprD4 and its NADPH-dependent reductase partner AprD3 20, 39, 40 . This C3 deoxygenation reaction does not occur on oxyapramycin but on the pseudodisaccharide substrate paromamine, demonstrating the two parallel biosynthetic pathways, which affords oxyapramycin and apramycin, respectively ( Figure 3) 20 .…”

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

“…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).…”

“…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.…”