title: 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.
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, The University of Alberta, Gunning-Lemieux Chemistry Centre, 11227 Saskatchewan Drive, Edmonton, AB T6G 2G2 (Canada).
authors.forename: Myles B
authors.initials: MB
authors.lastname: Poulin
authors.forename: Yun
authors.initials: Y
authors.lastname: Shi
authors.forename: Carla
authors.initials: C
authors.lastname: Protsko
authors.forename: Sean A
authors.initials: SA
authors.lastname: Dalrymple
authors.forename: David A R
authors.initials: DA
authors.lastname: Sanders
authors.forename: B Mario
authors.initials: BM
authors.lastname: Pinto
authors.forename: Todd L
authors.initials: TL
authors.lastname: Lowary
mesh_terms: D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D002169:Campylobacter Infections / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D016123:Campylobacter jejuni / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000737:chemistry / Q000378:metabolism; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014531:Uridine Diphosphate Galactose; D014536:Uridine Diphosphate N-Acetylgalactosamine; D001120:Arginine; D019751:Intramolecular Transferases
keywords: CORCEMA-ST calculations; NMR spectroscopy; X-ray crystallography; carbohydrates; molecular dynamics; pyranose-furanose mutase