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-beta-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 UDPgalactose as glycosyl donors and several nucleosides as acceptors. Kinetic analysis revealed that AvpGT is most specific for UDPglucose kcat/KMapp= (1.1 +/- 0.3) x 105 M-1 center dot s-1 as the glycosyl donor and tubercidin kcat/KMapp = (5.3 +/- 1.8) x 104 M-1 center dot s-1 as the glycosyl acceptor. NMR spectroscopic analysis revealed the product of this reaction to be 3 '-O-beta-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.
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