Characterization of uridine diphosphate-sugar pyrophosphorylase from Populus deltoids

Nucleotide sugars serve as sugar donors for the biosynthesis of various cell components including cell wall, glycoproteins, and small molecules. Among them, uridine diphosphate (UDP)-glucose is one of main nucleotide sugars that serve as a substrate for the synthesis of other UDP-sugars. UDP-sugar pyrophosphorylase (USPase) mediates the formation of UDP-glucose from uridine triphosphate (UTP) and glucose-1-phosphate. A USPase, PdUSPase was cloned from Populus deltoids and expressed in Escherichia coli as glutathione Stransferase fusion protein. The purified recombinant PdUSPase catalyzed the reaction for the formation of UDP-glucose from glucose-1-phosphate and UTP, and for the formation of UDP-galactose from galactose-1-phosphate and UTP. However, the enzyme did not show any activity toward mannose-1-phosphate and UTP. These results indicate that PdUSPase belonging in UGPase A in phylogenetic analysis is the first UDP-glucose synthesizing enzyme showing a discrepancy between phylogenetic analysis and substrate range. E. coli complementation was also carried out to confirm the function of PdUSPase using E. coligalU mutant, which was mutated in UTP glucose-1-phosphate uridyltransferase. The galU mutant was transformed with the PdUGTase gene and a flavonoid glucosyl-transferase gene, AtUGT78D2. The resulting transformant was able to convert quercetin into quercetin 3-O-glucose similarly to that by the wild type E. coli strain harboring AtUGT78D2. These results indicated that PdUSPase catalyzed the formation of UDP-glucose from UTP and glucose-1-phosphate.