Characterization of a bifunctional pyranose-furanose mutase from Campylobacter jejuni 11168.
UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-d-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.
Poulin
Myles B
MB
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
Nothaft
Harald
H
Hug
Isabelle
I
Feldman
Mario F
MF
Szymanski
Christine M
CM
Lowary
Todd L
TL
D000483:Alleles; D001120:Arginine / Q000378:metabolism; D055162:Biocatalysis; D001681:Biological Assay; D016123:Campylobacter jejuni / Q000201:enzymology*; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010080:Oxazoles / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism
D050505:Mutant Proteins; D010080:Oxazoles; D011135:Polysaccharides, Bacterial; D014536:Uridine Diphosphate N-Acetylgalactosamine; D001120:Arginine; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase