Biosynthesis of the antibiotic nucleocidin in Streptomyces calvus

Abstract

Fluorometabolites are a group of rare plant or bacterial natural products with around ten members. A most interesting organofluorine natural product is nucleocidin. Nucleocidin is an antibiotic isolated from Streptomyces calvus in 1957. The biosynthesis of nucleocidin involves a novel fluorination enzyme that is responsible for the 4’-C-F bond formation.

A reproducible production of nucleocidin was established from a commercial strain, S. calvus T-3018, provided by Pfizer. Isotopic labelling studies with different labelled glycerols were carried out. These were (1𝘙,2𝘙)-[²H₁]-, (1S,2𝘙)-[²H₁]-and [²H₅]- glycerols, all of which were successfully fed to S. calvus, demonstrates that both of the pro-𝘙 hydrogens become incorporated into the C5’ position of nucleocidin.

The structures of two novel 4’-fluoro-3’-O-β-glucosylated metabolites produced by S. calvus were elucidated. The glucosylation and deglucosylation of these metabolites catalysed by glucosyltransferase (NucGT) and β-glucosidase (NucBGS) enzymes were explored. The deglucosylation of the novel fluorometabolites produced nucleocidin and 4’-fluoroadenosine. The close location of the genes responsible for these enzymes further validates the biosynthetic gene cluster for nucleocidin biosynthesis.

The non-fluorinated counterparts of all three fluorometabolites were also isolated from the S. calvus culture. They were used to explore the fluorination with CFE and resting cells of S. calvus.

The novel glucosyltransferase (NucGT) that is responsible for the glucosylation in S. calvus was over-expressed and assayed. It is the first glucosyltransferase able to carry out glucosylation at the 3’-O position of a nucleoside. The kinetic parameters of different substrates with NucGT were determined, and the enzyme specificity was explored.