X-ray Crystallography Reveals a Reduced Substrate Complex of UDP-Galactopyranose Mutase Poised for Covalent Catalysis by Flavin

The flavoenzyme uridine 5'-diphosphate galactopyranose mutase (UGM or Glf) catalyzes the interconversion of UDP-galactopyra nose and UDP-galactofuranose. The latter Is a key building block for cell wall construction in numerous pathogens, including Mycobacterium tuberculosis. Mechanistic studies of UGM suggested a novel role for the flavin, and we previously provided evidence that the catalytic mechanism proceeds through a covalent flavin-galactose iminium. Here, we describe 2.3 and 2.5 resolution X-ray crystal structures of the substrate-bound enzyme in oxidized and reduced forms, respectively. In the latter, Cl of the substrate is 3.6 angstrom from the nucleophilic flavin N5 position. This orientation is consistent with covalent catalysis by flavin.