UDP-galactopyranose mutase has a novel structure and mechanism

Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the D-galac tofuranose (Galf) residues found in bacterial and parasitic cell walls, inc luding those of many pathogens, such as Mycobacterium tuberculosis and Tryp anosoma cruzi. UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase). The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a v iable therapeutic target. The mechanism by which mutase achieves the unprec edented ring contraction of a nonreducing sugar is unclear. We have solved the crystal structure of Escherichia coli mutase to 2.4 Angstrom resolution . The novel structure shows that the flavin nucleotide is located in a clef t lined with conserved residues. Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the subst rate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD. Assay results establish that the enzyme is active only when flavin is reduced. W e conclude that mutase most likely functions by transient reduction of subs trate.