title: Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease.
abstract: UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose (Galf). Galf is found in several pathogenic organisms, including the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Galf) is important for virulence and is not present in humans, making its biosynthetic pathway an attractive target for the development of new drugs against T. cruzi. Although UGMs catalyze a non-redox reaction, the flavin must be in the reduced state for activity and the exact role of the flavin in this reaction is controversial. The kinetic and chemical mechanism of TcUGM was probed using steady state kinetics, trapping of reaction intermediates, rapid reaction kinetics, and fluorescence anisotropy. It was shown for the first time that NADPH is an effective redox partner of TcUGM. The substrate, UDP-galactopyranose, protects the enzyme from reacting with molecular oxygen allowing TcUGM to turnover ∼1000 times for every NADPH oxidized. Spectral changes consistent with a flavin iminium ion, without the formation of a flavin semiquinone, were observed under rapid reaction conditions. These data support the proposal of the flavin acting as a nucleophile. In support of this role, a flavin-galactose adduct was isolated and characterized. A detailed kinetic and chemical mechanism for the unique non-redox reaction of UGM is presented.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, United States of America.
authors.forename: Michelle
authors.initials: M
authors.lastname: Oppenheimer
authors.forename: Ana Lisa
authors.initials: AL
authors.lastname: Valenciano
authors.forename: Karina
authors.initials: K
authors.lastname: Kizjakina
authors.forename: Jun
authors.initials: J
authors.lastname: Qi
authors.forename: Pablo
authors.initials: P
authors.lastname: Sobrado
mesh_terms: D014355:Chagas Disease; D005415:Flavins / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D005415:Flavins; C104136:uridine diphosphate galactofuranose; D009249:NADP; D014530:Uridine Diphosphate; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase; D005690:Galactose