Simultaneous one-pot multienzymatic synthesis and purification of Uridine-5′-Diphosphate-α-D-glucuronide and Uridine-5′-Diphosphate-α-d-xylose

Glucuronosyltransferases and xylosyltransferases are glycosyltransferases (GT) that are involved in a wide range of biological processes, including xenobiotic metabolism or proteoglycan biosynthesis and require as substrates activated sugar donors, respectively UDP-α-D-glucuronide (UDP-GlcA) and UDP-α-d-xylose (UDP-Xyl). As both nucleotide sugars are hardly available, we have developed a one-pot multienzymatic synthesis to produce sequentially UDP-GlcA and UDP-Xyl. This methodology involves the production of UDP-GlcA for glucuronic acid, coupling glucuronokinase and UDP-sugar pyrophosphorylase activities, followed by UDP-GlcA decarboxylation by UDP-Xylose Synthase. Each of these UDP-sugars can then be simultaneously purified and isolated from the unique reaction mixture, providing an efficient production methodology of these rare UDP-sugars for subsequent enzymatic studies of corresponding GT.