Comparing substrate specificity of two UDP-sugar pyrophosphorylases and efficient one-pot enzymatic synthesis of UDP-GlcA and UDP-GalA

•UDP-sugar pyrophosphorylase (AtUSP) from Arabidopsis thaliana showed significant activity towards both GlcA-1-P and GalA-1-P.•UDP-sugar pyrophosphorylase (BiUSP) from Bifidobacterium infantis showed significant activity towards GlcA-1-P.•Both USPs showed broad substrate specificity towards various sugar-1-phosphates.•Both AtUSP an BiUSP were expressed and purified in high yields (over 40 mg per L).•A one-pot three-enzyme approach was applied efficiently for hundreds mgs scale of UDP-GlcA and UDP-GalA.

Uridine 5′-diphosphate-glucuronic acid (UDP-GlcA) and UDP-galacturonic acid (UDP-GalA), the unique carboxylic acid-formed sugar nucleotides, are key precursors involved in the biosynthesis of numerous cell components. Limited availability of those components has been hindering the development of efficient ways towards facile synthesis of bioactive glycans such as glycosaminoglycans. In current study, we biochemically characterized two UDP-sugar pyrophosphorylases from Arabidopsis thaliana (AtUSP) and Bifidobacterium infantis ATCC15697 (BiUSP), and compared their activities towards a panel of sugar-1-phosphates and derivatives. Both enzymes showed significant pyrophosphorylation activities towards GlcA-1-phosphate, and AtUSP also exhibited comparable activity towards GalA-1-phosphate. By combining with monosaccharide-1-phosphate kinases, we have developed an efficient and facile one-pot three-enzyme approach to quickly obtain hundreds milligrams of UDP-GlcA and UDP-GalA.

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