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

Abstract

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 UDPgalactose as glycosyl donors and several nucleosides as acceptors. Kinetic analysis revealed that AvpGT is most specific for UDPglucose [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.

3’-O-β-Glycosylation of nucleoside analogues using a promiscuous bacterial glycosyltransferase

“…Only two nucleoside specific GTs have been identified and examined in detail: NucGT from S. calvus, 22,23 and AvpGT from Streptomyces sp. AVP053U2.…”

“…AVP053U2. 23 Both enzymes are members of the GT2 family, 24 whereby they catalyse glycosyl transfer through an inverting mechanism. Both NucGT and AvpGT share a high degree of sequence similarity, sharing 66% sequence identity.…”

“…Both NucGT and AvpGT share a high degree of sequence similarity, sharing 66% sequence identity. 23 AvpGT was selected, expressed and purified based on previously published protocols. 23 In the host species, AvpGT catalyses the 3'-O-glucosylation of tubercidin (7-deazadenosine, 2), a potent antimycobacterial and antineoplastic agent produced by various species of Streptomyces.…”

Purine nucleoside antibiotics: recent synthetic advances harnessing chemistry and biology

“…Furthermore, the identication of new metabolic enzymes from within nucleocidin biosynthesis provides opportunities to explore such derivatives and related systems, such as the 7deazapurine tubercidin. 70,71 As our ability to make mimetic protein structures progresses, an integration of synthetic biology within classical purine nucleoside chemical synthesis will be transformative. 72 Entering a post-antibiotic era predicates a need to develop new therapeutics.…”

“…Furthermore, the identification of new metabolic enzymes from within nucleocidin biosynthesis provides opportunities to explore such derivatives and related systems, such as the 7-deazapurine tubercidin. 70,71…”