The flavoenzyme uridine 5′-diphosphate (UDP)-galactopyranose mutase (UGM) catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). The latter is an essential precursor to the cell wall arabinogalactan of Mycobacterium tuberculosis. The catalytic mechanism for this enzyme had not been elucidated. Here, we provide evidence for a mechanism in which the flavin cofactor assumes a new role. Specifically, the N5 of the reduced anionic flavin cofactor captures the anomeric position of the galactose residue with release of UDP. Interconversion of the isomers occurs via a flavin-derived iminium ion. To trap this putative intermediate, we treated UGM with radiolabeled UDP-Galp and sodium cyanoborohydride; a radiolabeled flavin-galactose adduct was obtained. Ultraviolet-visible spectroscopy and mass spectrometry indicate that this product is an N5-alkyl flavin. We anticipate that the clarification of the catalytic mechanism for UGM will facilitate the development of anti-mycobacterial agents.
Bibliographical noteFunding Information:
We acknowledge R. Raines, P. Ludden (University of Wisconsin Madison) and the W. M. Keck Center for use of equipment. We thank R. Raines, A. Eschenmoser, W. Cleland, G. Reed, M. Wolfe, M. Vestling, and R. Derda for helpful conversations. This research was supported by the US National Science Foundation (CHE9357093), the US National Institutes of Health (NIH) (GM49975) and the Mizutani Foundation for Glycoscience. M.S.H., E.E.C. and T.D.G. were supported by the NIH Biotechnology Training Program (GM 08349). M.S.H. was supported by an NIH predoctoral fellowship (GM 18750).