Discovery of 3'- O -β-Glucosyltubercidin and the Nucleoside Specific Glycosyltransferase AvpGT through Genome Mining.

A genome mining approach was used to identify a hybrid tubercidin-nucleocidin biosynthetic gene cluster (BGC) in Streptomyces sp. AVP053U2. Analysis of culture extracts by liquid chromatography-mass spectrometry revealed the presence of a glucosylated tubercidin derivative. A gene, avpGT , was identified within the hybrid cluster that has homology to the glucosyltransferase that is responsible for 3'- O -β-glucosylation of the fluorinated natural product nucleocidin. AvpGT was heterologously expressed and purified from Escherichia coli for in vitro characterization. AvpGT is active toward UDP-glucose and UDP-galactose as glycosyl donors and several nucleosides as acceptors. Kinetic analysis revealed that AvpGT is most specific for UDP-glucose [ k cat / K M app = (1.1 ± 0.3) × 10 5 M -1 ·s -1 ] as the glycosyl donor and tubercidin [ k cat / K M app = (5.3 ± 1.8) × 10 4 M -1 ·s -1 ] as the glycosyl acceptor. NMR spectroscopic analysis revealed the product of this reaction to be 3'- O -β-glucopyranosyl tubercidin. A sequence analysis of AvpGT reveals a family of nucleoside-specific GTs, which may be used as markers of BGCs that produce glycosylated nucleosides.