A unique catalytic mechanism for UDP-galactopyranose mutase.
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.
Soltero-Higgin
Michelle
M
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Carlson
Erin E
EE
Gruber
Todd D
TD
Kiessling
Laura L
LL
D002384:Catalysis; D005415:Flavins; D007097:Imines; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000378:metabolism; D007536:Isomerism; D013058:Mass Spectrometry; D013056:Spectrophotometry, Ultraviolet; D014530:Uridine Diphosphate / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000737:chemistry / Q000378:metabolism*
D005415:Flavins; D007097:Imines; D014531:Uridine Diphosphate Galactose; D014530:Uridine Diphosphate; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase