Mechanistic and structural studies of eukaryotic UDP-galactopyranose mutases

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): 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. )