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

Abstract

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.

Keywords: Synthesis; UDP-GalA; UDP-GlcA; UDP-sugar pyrophosphorylase.

Figure 1

De novo and salvage biosynthetic pathway of UDP-GlcA and UDP-GalA. A), Glucuronokinase (GlcAK); B) UDP-sugar pyrophosphorylase (USP); C) UDP-glucose 6-dehydrogenase; D) Phosphoglucomutase; E) UTP: glucose-1-phosphate uridylyltransferase (GalU); F) UDP-Glc 4’-epimerase (GalE); G) UDP-galactose 6-dehydrogenase; H) galacturonokinase (GalAK); I) UDP-galacturonate pyrophosphorylase.

Figure 2

SDS-PAGE analysis of purified enzymes. Lanes: 1, AtGlcAK; 2, AtUSP; 3, BiGalK; 4, BiUSP.

Figure 3

CE Profiles of compound standards, and BiUSP/AtUSP catalyzed reactions. (A) 0.5 mM UTP; (B) 0.5 mM UDP-GlcA; (C) 0.5 mM of UDP-GalA; (D) BiUSP catalyzed generation of UDP-GlcA; (E) AtUSP catalyzed generation of UDP-GlcA; (F) AtUSP catalyzed generation of UDP-GalA. For each reaction, 0.8 mM sugar-1-P, 1 mM UTP, and 1 µg enzyme was used in a total 50 µL system, reactions were proceeded at 37 °C for 20 min. UTP has a retention time of 21.7 ± 0.1 min, UDP-GlcA has a retention time of 18.2 ± 0.1 min, and UDP-GalA has a retention time of 16.6 ± 0.1 min.

Figure 4

Time course for UDP-GlcA and UDP-GalA reactions. Reactions contains 1 mM UTP, 40 ng of At USP or BiUSP, and: 1, AtUSP and 0.05 mM GlcA-1-P; 2, AtUSP and 0.8 mM GlcA-1-P; 3, AtUSP and 0.05 mM GalA-1-P; 4, AtUSP and 0.8 mM GalA-1-P; 5, BiUSP and 0.05 mM GlcA-1-P; 6, BiUSP and 0.8 mM GlcA-1-P.

Figure 5

One-pot three-enzyme synthesis of UDP-GlcA and UDP-GalA. Enzymes used: AtGlcAK: Glucuronokinase from A. thaliana; BiGalK, Galactokinase from B. infantis ATCC15697; AtUSP, UDP-sugar pyrophosphorylase from A. thaliana; EcPpA, Pyrophosphatase from E. coli.