Galactofuranose (Galf) is an important building block of the cell wall of pathogenic fungi and a major component of the cell surface glycoconjugated structures (sugar coat) of protozoan parasites. Because of the importance of Galf-containing molecules for host-specific cell recognition, growth, and pathogenesis, and the absence of this unusual sugar in humans, Galf biosynthetic enzymes are attractive targets for the development of new antimicrobial agents. The flavoenzyme UDP-galactopyranose mutase (UGM) plays a central role in Galf biosynthesis by catalyzing the conversion of UDP-galactopyranose to UDP-Galf. Deletion of the UGM gene results in severely attenuated virulence of the fungal pathogen Aspergillus fumigatus and the protozoan parasite Leishmania major, suggesting UGM as a promising drug design target. In addition, UGM is fundamentally interesting because the enzyme neither oxidizes nor reduces the substrate, which is unusual among flavoenzymes. Studies of bacterial UGMs have shown that the reduced flavin is necessary for catalysis, but the role that the flavin plays during the catalytic cycle remains controversial. Here, we propose the first studies of the catalytic mechanism and three-dimensional structure of eukaryotic UGMs, using the enzymes from A. fumigatus and Trypanosoma cruzi as prototypes from fungal and protozoan parasites, respectively. Key preliminary results include the production of active recombinant enzyme and the growth of preliminary crystals.
Two aims are proposed: 1. Determine the role of the flavin cofactor in the chemical mechanism of eukaryotic UGMs. Experiments proposed include rapid reaction kinetic spectroscopic analyses, characterization of the redox potential and pH profiles, testing potential alternative substrates and inhibitors, and identifying redox partners. 2. Determine the three-dimensional structures of eukaryotic UGMs. Structures of UGMs in the oxidized, reduced, and ligand-bound conformations will be solved using X-ray crystallography and small-angle X-ray scattering. Successful completion of these aims will provide a platform for the future design of structure- and mechanism- based inhibitors of UGMs, which could serve as lead compounds for the development of chemotherapeutics for the treatment of fungal infections and neglected diseases such as Chagas disease. )
UDP-galactopyranose mutase (UGM) catalyzes a key step in the biosynthesis of galactofuranose (Galf), an essential sugar molecule found in pathogenic fungi and disease-causing protozoan parasites. The proposed research will create new knowledge about the catalytic mechanism and three-dimensional structure of UGM from the fungus Aspergillus fumigatus and the causative agent of Chagas disease, the parasite Trypanosoma cruzi. This information will aid the development of antimicrobial agents targeting these important human pathogens.
|Tanner, John J; Boechi, Leonardo; Andrew McCammon, J et al. (2014) Structure, mechanism, and dynamics of UDP-galactopyranose mutase. Arch Biochem Biophys 544:128-41|
|Boechi, Leonardo; de Oliveira, Cesar Augusto F; Da Fonseca, Isabel et al. (2013) Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design. Protein Sci 22:1490-501|
|Kizjakina, Karina; Tanner, John J; Sobrado, Pablo (2013) Targeting UDP-galactopyranose mutases from eukaryotic human pathogens. Curr Pharm Des 19:2561-73|
|Komachi, Yuji; Hatakeyama, Shintaro; Motomatsu, Haruka et al. (2013) GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol Microbiol 90:1054-73|
|Fonseca, Isabel O; Kizjakina, Karina; Sobrado, Pablo (2013) UDP-galactopyranose mutases from Leishmania species that cause visceral and cutaneous leishmaniasis. Arch Biochem Biophys 538:103-10|
|Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle et al. (2012) Crystal structures and small-angle x-ray scattering analysis of UDP-galactopyranose mutase from the pathogenic fungus Aspergillus fumigatus. J Biol Chem 287:9041-51|
|Oppenheimer, Michelle; Valenciano, Ana Lisa; Kizjakina, Karina et al. (2012) Chemical mechanism of UDP-galactopyranose mutase from Trypanosoma cruzi: a potential drug target against Chagas' disease. PLoS One 7:e32918|