Structural determination of archaeal UDP‐N‐acetylglucosamine 4‐epimerase from Methanobrevibacter ruminantium M1 in complex with the bacterial cell wall intermediate UDP‐N‐acetylmuramic acid

Abstract

The crystal structure of UDP-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase;WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N-terminal Rossmann-fold for nucleotide binding and an active site positioned in the C-terminus. UDP-GlcNAc 4-epimerase is a member of the short-chain dehydrogenases/reductases superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4-epimerases. The protein was co-crystallized with coenzyme NADH and UDP-N-acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non-native UDP sugar amongst archaea and was most likely incorporated from the E. coli expression host during purification of the recombinant enzyme. K E Y W O R D S 4-epimerase, EPZ, Methanobrevibacter, Pseudomurein, UDP-GlcNAc 4-epimerase, UDP-Nacetylglucosamine, UDP-N-acetylmuramic acid, WbpP

Enzymatic C4‐Epimerization of UDP‐Glucuronic Acid: Precisely Steered Rotation of a Transient 4‐Keto Intermediate for an Inverted Reaction without Decarboxylation

“…UDP‐galactose 4‐epimerase, UDP‐ N ‐acetyl‐glucosamine 4‐epimerase, and others) and so involve 4‐keto intermediates chemically less vulnerable than that of UGAepi, are thought (based on the static evidence of crystal structures) to make do in their catalysis with just rigid‐body rotations (Figure S31). [ 3g , 3h , 17 , 18 , 19 , 20 ] Our results suggest the UGAepi‐specific strategies used to prevent decarboxylation of the 4‐ketohexuronic acid. First, the rotational endpoints I and I ROT manifest conformational sampling by the enzyme to implement stereoelectronic control.…”

“…SDR epimerases that use hexose/pentose nucleotide substrates (e.g. UDP‐galactose 4‐epimerase, UDP‐ N ‐acetyl‐glucosamine 4‐epimerase, and others) and so involve 4‐keto intermediates chemically less vulnerable than that of UGAepi, are thought (based on the static evidence of crystal structures) to make do in their catalysis with just rigid‐body rotations (Figure S31) [3g,h, 17–20] . Our results suggest the UGAepi‐specific strategies used to prevent decarboxylation of the 4‐ketohexuronic acid.…”

Structural Analysis of Cj1427, an Essential NAD-Dependent Dehydrogenase for the Biosynthesis of the Heptose Residues in the Capsular Polysaccharides of Campylobacter jejuni

“…Thus, no recycling mechanisms are required. The top matches include UDP- N -acetylglucosamine 4-epimerase from Methanobrevibacter ruminantium M1 and PAL from Bacillus thuringiensis . , Shown in Figure a is a superposition of the Cj1427 and M. ruminantium M1 epimerase subunits. The α-carbons for these two models superimpose with a root-mean-square deviation of ∼2 Å.…”

Glycolipid composition of the heterocyst envelope of Anabaena sp. PCC 7120 is crucial for diazotrophic growth and relies on the UDP‐galactose 4‐epimerase HgdA

“…They take part in important metabolic processes, for example, UDP‐galactose 4‐epimerase participates in the Leloir pathway, in which it converts UDP‐galactose to UDP‐glucose (Beerens, Soetaert, & Desmet, ; Maxwell, ; Wilson & Hogness, ). Epimerases mainly constitute dimers, however, other oligomeric states can also be found; the structures of some of these proteins have been resolved (Bauer, Rayment, Frey, & Holden, ; Carbone, Schofield, Sang, Sutherland‐Smith, & Ronimus, ; Deacon, Ni, Coleman, & Ealick, ; Giraud, Leonard, Field, Berlind, & Naismith, ). In the present study, we investigated the role of the putative epimerase HgdA in Anabaena sp.…”