Characterization of a bifunctional pyranose-furanose mutase from Campylobacter jejuni 11168

Myles B. Poulin, Harald Nothaft, Isabelle Hug, Mario F. Feldman, Christine M. Szymanski, Todd L. Lowary

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)493-501
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number1
DOIs
StatePublished - Jan 1 2010

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