UDP-galactopyranose mutase (UGM) is a flavoenzyme which catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDPGalf). UDP-Galf is the active precursor of Galf residues. Glycoconjugates of Galf residues are found in the cell wall of bacteria and on the cell surface of higher eukaryotes. Galf residues have not been found in humans and the fact that they are essential for the growth of pathogenic bacteria makes UGM a potential antibacterial target. In the present study, crystal structures of UGM from Deinocococcus radiodurans (drUGM) in complex with substrate (UDP-Galp) were determined. UDP-Galp is buried in the active site and bound in a U-shaped conformation. The binding mode and active site interactions of UDP-Galp are consistent with the previous biochemical and mechanistic studies. The mobile loops in the substrate complex structures exist in a closed conformation and Arg198 on one of the mobile loops stabilizes the phosphate groups of the substrate. The anomeric carbon of galactose is 2.8 Å from the N5 of FAD (in the reduced complex) favorable to form FAD-galactosyl adduct. In addition to substrate complex structures, the crystal structures of drUGM in complex with UDP, UMP, and UDP-Glc have been determined. The mobile loops in all these complexes exist in a closed conformation. Inhibitors for UGM were identified by ligand-based and structure-based methods. The phosphonate analog of UDP-Galp (GCP) showed only weak inhibition against various bacterial UGMs. The structure of drUGM in complex with GCP provided a basis for its inhibitory activity. Poor stabilization of the phosphate groups by conserved arginines (Arg198 and Arg305) and altered sugar binding mode account for its activity. Novel indole-based (LQ1, LQ6 and LQ10) inhibitors of UGM were identified through structure-based virtual screening (SBVS) of a chemical library. Inhibition studies also allowed the identification of an active site aspartic acid that plays role in inhibitor binding. The structural studies on drUGM provided a basis for understanding substrate binding to UGM. In vitro enzyme inhibition studies allowed the identification of novel indole-based inhibitors. The structural and inhibition studies reported here enhance the understanding of UGM-ligand interactions and will assist in the development of more potent inhibitors of UGM.

UDP-galactopyranose mutase (UGM) is a flavoenzyme which catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDPGalf). UDP-Galf is the active precursor of Galf residues. Glycoconjugates of Galf residues are found in the cell wall of bacteria and on the cell surface of higher eukaryotes. Galf residues have not been found in humans and the fact that they are essential for the growth of pathogenic bacteria makes UGM a potential antibacterial target. In the present study, crystal structures of UGM from Deinocococcus radiodurans (drUGM) in complex with substrate (UDP-Galp) were determined. UDP-Galp is buried in the active site and bound in a U-shaped conformation. The binding mode and active site interactions of UDP-Galp are consistent with the previous biochemical and mechanistic studies. The mobile loops in the substrate complex structures exist in a closed conformation and Arg198 on one of the mobile loops stabilizes the phosphate groups of the substrate. The anomeric carbon of galactose is 2.8 Å from the N5 of FAD (in the reduced complex) favorable to form FAD-galactosyl adduct. In addition to substrate complex structures, the crystal structures of drUGM in complex with UDP, UMP, and UDP-Glc have been determined. The mobile loops in all these complexes exist in a closed conformation. Inhibitors for UGM were identified by ligand-based and structure-based methods. The phosphonate analog of UDP-Galp (GCP) showed only weak inhibition against various bacterial UGMs. The structure of drUGM in complex with GCP provided a basis for its inhibitory activity. Poor stabilization of the phosphate groups by conserved arginines (Arg198 and Arg305) and altered sugar binding mode account for its activity. Novel indole-based (LQ1, LQ6 and LQ10) inhibitors of UGM were identified through structure-based virtual screening (SBVS) of a chemical library. Inhibition studies also allowed the identification of an active site aspartic acid that plays role in inhibitor binding. The structural studies on drUGM provided a basis for understanding substrate binding to UGM. In vitro enzyme inhibition studies allowed the identification of novel indole-based inhibitors. The structural and inhibition studies reported here enhance the understanding of UGM-ligand interactions and will assist in the development of more potent inhibitors of UGM.

Galactofuranose (Galf) is the five membered ring form of galactose. It is found on the cell wall and surface of many pathogens including Mycobacterium tuberculosis, Aspergillus fumigatus, Leishmania major, and Trypanosoma cruzi. Galf has been implicated in pathogenesis in these organisms; thus the biosynthetic pathway of Galf is a target for drug design. Galf is synthesized by the enzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). Solving the mechanism and structure of UGMs will aid in the development of specific inhibitors against these enzymes. Herein we present the detailed functional analysis of UGMs from A. fumigatus, T. cruzi, and L. major. The mechamism and structure these eukaryotic UGMs were examined by steady-state kinetics, rapid-reaction kinetics, trapping of reaction intermediates, fluorescence anisotropy, and X-ray crystallography. The mechanism first involves reduction of the required flavin by NADPH, followed by UDP-Galp binding and subsequent SN2 attack by the flavin on galactose displacing UDP to form a flavin N5-C1 galactose adduct. Next, the galactose ring opens forming an iminium ion allowing isomerization to occur. Lastly, the product is released and UGM is available to bind another substrate or be reoxidized by molecular oxygen. The three-dimensional structure of A. fumigatus UGM was solved using X-ray crystallography in four conformations: oxidized in complex with sulfate ions, reduced, reduced in complex with UDP, and reduced in complex with UDP-Galp, giving valuable information on the unique features of eukaryotic UGMs including features important for oligomerization and for substrate binding. The novel mechanism and structure provide valuable information for the development of specific inhibitors of eukaryotic UGMs. / Ph. D.

Made available in DSpace on 2015-03-03T14:00:59Z (GMT). No. of bitstreams: 1 DiegoAS_DISSERT.pdf: 1474988 bytes, checksum: 09ed96a182fab36b0c95800c391aded7 (MD5) Previous issue date: 2010-04-14 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Os polissacar?deos sulfatados comp?em um grupo complexo de macromol?culas com uma gama de importantes atividades biol?gicas, incluindo atividade antiviral, anticoagulante, antiproliferativa, antitumoral e antioxidante. Esses pol?meros ani?nicos s?o amplamente distribu?dos em tecidos de vertebrados e algas. As algas marinhas s?o as fontes, n?o-mam?feras, mais abundantes de polissacar?deos sulfatados na natureza. Os mais conhecidos extra?dos de algas vermelhas s?o homogalactanas, chamadas de carragenanas, e de algas marrons s?o homo ou heteropolissacar?deos, chamadas de fucanas e fucoidanas, respectivamente, e os polissacar?deos sulfatados de algas verdes s?o compostas por polissacar?deos contendo galactose, glicose, arabinose e/ou ?cido glucur?nico. H? poucos estudos descrevendo a presen?a de polissacar?deos sulfatados em algas verdes. No presente estudo, o extrato total de polissacar?deos da alga verde Codium isthmocladum foi obtido atrav?s de digest?o proteol?tica, e fracionado por precipita??o sequencial com acetona resultando em cinco fra??es (F0,3V; F0,5V; F0,7V; F0,9V e F1,2V). O teste de atividade anticoagulante de aPTT mostrou que F0,5V desempenhou alta atividade anticoagulante. Esta foi, posteriormente, fracionada por cromatografia de troca-i?nica em sete polissacar?deos: F0,3M; F0,5M; F0,7M; F1,0M; F1,5M; F2,0M e F3,0M. Os polissacar?deos elu?dos com F2,0M e F3,0M migraram como um ?nico componente eletrofor?tico. N?o foi detectada contamina??o prot?ica. An?lises qu?micas mostraram que F2,0M e F3,0M s?o compostas por galactose. Esses polissacar?deos foram submetidos ao teste de aPTT, e mostraram alta atividade anticoagulante com atividade maior que Clexane?. Ambas apresentaram um efeito dose-dependente. An?lises estruturais de F2,0M e F3,0M mostraram que elas s?o homopol?meros ramificados constitu?dos de - D-Galactopiranoses unidas por liga??es b-1,6 (predominante) e b-1,3. Alguns res?duos de galactose apresentam sulfata??o em C4 ou em C6, e algumas galactoses em posi??o n?o redutora apresentam piruvata??o na forma de 3,4- O-(1?-carboxi)etilideno, cetal c?clico com cinco membro