Two 3'- O-β-glucosylated nucleoside fluorometabolites related to nucleocidin in Streptomyces calvus

Abstract

The antibiotic nucleocidin is a product of the soil bacterium Streptomyces calvus T-3018. It is among the very rare fluorine containing natural products but is distinct from the other fluorometabolites in that it is not biosynthesised from 5'-fluorodeoxyadenosine via the fluorinase. It seems to have a unique enzymatic fluorination process. We disclose here the structures of two 4'-fluoro-3'-O-β-glucosylated metabolites (F-Mets I and II) which appear and then disappear before nucleocidin production in batch cultures of S. calvus. Full genome sequencing of S. calvus T-3018 and an analysis of the putative biosynthetic gene cluster for nucleocidin identified UDP-glucose dependent glucosyl transferase (nucGT) and glucosidase (nucGS) genes within the cluster. We demonstrate that these genes express enzymes that have the capacity to attach and remove glucose from the 3'-O-position of adenosine analogues. In the case of F-Met II, deglucosylation with the NucGS glucosidase generates nucleocidin suggesting a role in its biosynthesis. Gene knockouts of nucGT abolished nucelocidin production.

Fig. 1. (A) Nucleoside antibiotics 1–3. (B) The fluorinated natural products 5–7 derive from the fluorinase via FDA 4, however this enzyme is not involved in nucleocidin 1 biosynthesis.

Fig. 2. ¹⁹F-NMR time course of nucleocidin 1 production in S. calvus showing the appearance and disappearance of F-Mets I and II over 11 days.

Fig. 3. Organisation of the putative nucleocidin biosynthetic gene cluster in S. calvus. A fuller annotation and a comparison with the ascamycin 2 antibiotic gene cluster, can be found in the ESI Fig. S36.

Fig. 4. Comparison of the initial rates (triplicates) of 8–10 with Nuc-GT. Rates 8 (), 9 () and 10 ().

Fig. 5. Synthesis route to 3′-O-β-gluco-adenosine 11.

Fig. 6. ¹⁹F{¹H}-NMR time course profiles of biotransformations of F-Mets I and II with β-glucosidase NucGS. (a) F-Met I is converted to 4′-fluoroadenosine 19. (b) F-Met II is converted to nucleocidin 1.

Fig. 7. Putative minimal pathway for nucleocidin 1 biosynthesis.