Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

Abstract

UDP-hexose 4-epimerases play a pivotal role in lipopolysaccharide (LPS) biosynthesis and Leloir pathway. These epimerases are classified into three groups based on whether they recognize nonacetylated UDP-hexoses (Group 1), both N-acetylated and nonacetylated UDPhexoses (Group 2) or only N-acetylated UDP-hexoses (Group 3). Although the catalysis has been investigated extensively, yet a definitive model rationalizing the substrate specificity of all the three groups on a common platform is largely lacking. In this work, we present the crystal structure of WbgU, a novel UDP-hexose 4-epimerase that belongs to the Group 3. WbgU is involved in biosynthetic pathway of the unusual glycan 2-deoxy-L-altruronic acid that is found in the LPS of the pathogen Pleisomonas shigelloides. A model that defines its substrate specificity is proposed on the basis of the active site architecture. Representatives from all the three groups are then compared to rationalize their substrate specificity. This investigation reveals that the Group 3 active site architecture is markedly different from the ''conserved scaffold'' of the Group 1 and the Group 2 epimerases and highlights the interactions potentially responsible for the origin of specificity of the Group 3 epimerases toward N-acetylated hexoses. This study provides a platform for further engineering of the UDP-hexose 4-epimerases, leads to a deeper understanding of the LPS biosynthesis and carbohydrate recognition by proteins. It may also have implications in development of novel antibiotics and more economic synthesis of UDP-GalNAc and downstream products such as carbohydrate based vaccines.

UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specificity

“…57 The availability of a second group 3 epimerase, the WbgU from Pleisomonas shigelloides, and its structure provided more information on the substrate specificity of these enzymes. 16 Despite the relatively low sequence identity among all three groups, the similarity of the enzymes' tertiary structures is striking with an overall rmsd of the multiple structure alignment being 1.08 Å and variation being most pronounced at the C-terminal end. The model of the '297e308 belt' was proposed to determine substrate specificity in group 3 members.…”

“…As a result, the S306Y mutation, which allows a switch from group 2 to group 1, forms steric clashes between the group 3 epimerases and their substrates, which results in the observed loss of activity. 16 The importance and flexibility of the hydrogen bond network was evidenced by multiple single mutants as well as their combined double, triple and quadruple mutants. 58 …”

“…Among these were epimerases active on UDP-Glc/UDP-Gal, 28 UDP-GlcNAc/UDP-GalNAc, 16 UDP-GlcUA/UDP-GalUA (uronic acid form), 17 UDP-L-Ara/UDP-Xyl (pentoses), 22 or multiple of these substrates. 13,14,23 As they are involved in different pathways and functions, such as protein glycosylation and production and secretion of virulence factors, these enzymes are possible targets for new therapeutics.…”

“…1 GalE and related enzymes display unusual enzymologic, chemical, and stereochemical properties; including irreversible binding of NAD and uridine nucleotide-induced activation of NAD, 21 which will be further addressed below. The UDP-sugars 4-epimerase family consists of epimerase active on UDP-Glc/UDP-Gal, N-acetylated forms (UDP-GlcNAc/UDP-GalNAc), 16 uronic acid forms (UDP-GlcUA/ UDP-GalUA) 17 as well as UDP-pentoses (UDP-L-Ara/UDP-Xyl) 22 or on combinations thereof. 13,14,23,24 Attempts have been made to elucidate the determinants for substrate specificity of UDP-sugars 4-epimerases as this can render information to use these epimerases as targets for new antibiotics and drugs.…”

“…14,15 Apart from non-modified sugars, also N-acetylated and carboxylated sugars are found in LPS. 16,17 Consequently, the corresponding UDP-sugars have to be made by the cells and GalE-like UDP-sugars 4-epimerases are used for this purpose. UDP-galactose 4-epimerase is also found to be important for the biosynthesis of other polysaccharide structures, such as capsular polysaccharide (CPS), 15 extracellular polysaccharide (EPS) from Streptococcus thermophiles 18 eone of the most widely used bacteria in the dairy industryeand proteoglycans (PGs) of articular chondrocytes.…”