Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase

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dc.contributor.authorQi, Junen
dc.contributor.authorOppenheimer, Michelleen
dc.contributor.authorSobrado, Pabloen
dc.contributor.departmentBiochemistryen
dc.contributor.departmentFralin Life Sciences Instituteen
dc.contributor.departmentCenter for Drug Discoveryen
dc.date.accessioned2017-09-18T09:51:33Zen
dc.date.available2017-09-18T09:51:33Zen
dc.date.issued2011-08-21en
dc.date.updated2017-09-18T09:51:33Zen
dc.description.abstractAspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with Kd values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z′ factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationJun Qi, Michelle Oppenheimer, and Pablo Sobrado, “Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase,” Enzyme Research, vol. 2011, Article ID 513905, 9 pages, 2011. doi:10.4061/2011/513905en
dc.identifier.doihttps://doi.org/10.4061/2011/513905en
dc.identifier.urihttp://hdl.handle.net/10919/79016en
dc.language.isoenen
dc.publisherHindawien
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.holderCopyright © 2011 Jun Qi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleFluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutaseen
dc.title.serialEnzyme Researchen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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