Specificity of a UDP‐GalNAc Pyranose–Furanose Mutase: A Potential Therapeutic Target for Campylobacter jejuni Infections

Abstract

Pyranose-furanose mutases are essential enzymes in the life cycle of a number of microorganisms, but are absent in mammalian systems, and hence represent novel targets for drug development. To date, all such mutases show preferential recognition of a single substrate (e.g., UDP-Gal). We report here the detailed structural characterization of the first bifunctional pyranose-furanose mutase, which recognizes both UDP-Gal and UDP-GalNAc. The enzyme under investigation (cjUNGM) is involved in the biosynthesis of capsular polysaccharides (CPSs) in Campylobacter jejuni 11168. These CPSs are known virulence factors that are required for adhesion and invasion of human epithelial cells. Using a combination of UV/visible spectroscopy, X-ray crystallography, saturation transfer difference NMR spectroscopy, molecular dynamics and CORCEMA-ST calculations, we have characterized the binding of the enzyme to both UDP-Galp and UDP-GalpNAc, and compared these interactions with those of a homologous monofunctional mutase enzyme from E. coli (ecUGM). These studies reveal that two arginines in cjUNGM, Arg59 and Arg168, play critical roles in the catalytic mechanism of the enzyme and in controlling its specificity to ultimately lead to a GalfNAc-containing CPS. In ecUGM, these arginines are replaced with histidine and lysine, respectively, and this results in an enzyme that is selective for UDP-Gal. We propose that these changes in amino acids allow C. jejuni 11168 to produce suitable quantities of the sugar nucleotide substrate required for the assembly of a CPS containing GalfNAc, which is essential for viability.

Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents

“…Structures of UGM homologues from two eukaryotic species ( Aspergillus fumigatus 43 and Trypanosoma cruzi 44 ) and five different bacterial species 6,21,45–48 have been determined. Because we were seeking antimycobacterial agents, we focused on structures of UGMs in the active conformation from bacterial species.…”

A Second, Druggable Binding Site in UDP‐Galactopyranose Mutase from Mycobacterium tuberculosis?

“…After equilibration, production MD simulations were conducted for 80 ns for each system without any constraints. This timescale was chosen based on available computational resources and previous work on UGM . Three replicate MD runs were performed for each system by varying the random seed for initial velocity generation.…”

Strategies toward protecting group-free glycosylation through selective activation of the anomeric center

“…One key step in the biosynthesis of these hexofuranoses is the isomerization of uridine 5′-diphospho (UDP)-pyranose to the corresponding furanosyl donor catalyzed by pyranose mutases. Shown is the transformation of UDP-Gal p to UDP-Gal f catalyzed by UDP-galactopyranose mutase (UGM) (Scheme 11) [44–45]. A more expedient access to hexofuranose analogs could have implications in the study of infection and potential treatments.…”