Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum

Young-Woo Nam,Shinya Fushinobu,Takatoshi Arakawa,Mamoru Nishimoto,Motomitsu Kitaoka

doi:10.1038/s41598-019-47591-w

Abstract

Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

Highlights

Bifidobacteria are anaerobic gram-positive bacteria belonging to the genus Bifidobacterium and are usually found in the gastrointestinal tract of humans and animals[1]
Group 1 enzymes preferentially catalyze the epimerization between UDP-Glc and UDP-Gal, group 2 enzymes do not show a preference for either UDP-Glc/UDP-Gal or UDP-GlcNAc/ UDP-GalNAc, and group 3 enzymes preferentially catalyze the epimerization between UDP-GlcNAc and UDP-GalNAc (WbpP) (Table 1)
We previously investigated biochemical characteristics of a paralog glycans. UDPglucose 4-epimerase (GalE) enzyme from the same organism, B. longum JCM1217 (BLLJ_1592 corresponding to BL1671 of B. longum NCC2705)[44], whose gene is not accompanied by other galactose metabolic genes

Summary

Introduction

Bifidobacteria are anaerobic gram-positive bacteria belonging to the genus Bifidobacterium and are usually found in the gastrointestinal tract of humans and animals[1]. The structural unit of lacto-N-biose I (Gal-β1,3-GlcNAc, LNB) is predominantly present in HMOs but not in milk of other mammals[11], and LNB has been shown to be the bifidus factor, which promotes growth of infant gut-associated bifidobacteria[12,13]. GalE catalyzes NAD+-dependent oxidoreductive interconversion of gluco- and galacto-hexoses (C4-epimerization) linked to UDP (Fig. 1) and generally plays a key role in the metabolism of galactose in various organisms[19,20,21]. Crystal structures of GalEs from Escherichia coli (eGalE)[22,23,24,25,26,27], Trypanosoma brucei (tGalE)[28], human (hGalE)[29,30,31], Pseudomonas aeruginosa (WbpP)[32], Pyrobaculum calidifontis[33], Aspergillus nidulans[34], and several others have been reported. We describe the crystal structures of bGalE and compare them with GalEs from other organisms

Methods

Results

Conclusion