UDP-galactopyranose mutase (EC 5.4.99.9; UGM), the product of the glf gene, is an
enzyme that catalyzes the conversion of uridine diphosphate galactopyranose (UDP-Galp) to
UDP-galactofuranose (UDP-Galf). UGM activity is found in bacteria, parasites and fungi,
however is absent in higher eukaryotes. This enzyme is essential for the viability of many
pathogenic organisms, such as Mycobacterium tuberculosis and Escherichia coli, due to the
broad distribution of Galf in crucial structures such as the cell wall or capsular polysaccharide.
Not surprisingly, galactofuranose biosynthesis has become an attractive antimicrobial target due
to the absence of these sugars in higher eukaryotes. The UGM homologue, UDP-Nacetylgalactopyranose
mutase (UNGM), was identified in Campylobacter jejuni 11168,
encoded for by the cj1439c gene. UNGM is known to function as a bifunctional mutase, which
catalyzes the reversible ring contraction between the pyranose-furanose forms of UDPgalactose
(UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc). UNGM is essential for
the virulence of C. jejuni, due to the incorporation of UDP-N-acetylgalactofuranose into the
capsular polysaccharide. We report the first structure of UNGM determined by X-ray
crystallography, to a resolution of 1.9 Å. Analysis of the dimeric, holoenzyme structure of
UNGM has identified that the cofactor flavin adenine dinucleotide is bound within each
monomer of the enzyme. Comparative analysis with UGM homologues has confirmed the
conserved active site residues involved in the binding of various substrates. Docking studies
suggest that UNGM binds its natural substrates in a productive binding mode for catalysis with
the flavin cofactor, which is consistent with the proposed mechanism for UNGM. The mobile
loops are essential for substrate binding, and we have identified that the conserved arginine
residue, Arg169, and the neighboring Arg168, function to stabilize the diphosphate region of
UDP, although not concurrently. The non-conserved arginine residue, Arg168, appeared to
favor the stabilization of N-acetylated sugars, which is in agreement with the enzyme’s higher
binding affinity for UDP-GalNAc over UDP-Gal by a factor of 0.9. We have also identified
that the active site Arg59 exists in two conformations in the structure of UNGM, with one
conformation directed toward the active site. Arg59 is 2.5 to 3.0 Å from the acetamido moiety
of GalNAc, which is favorable for stabilization and is believed to confer specificity for this
substrate.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2012-11-806 |
| Date | 2012 November 1900 |
| Contributors | Sanders, David A. |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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