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Enzyme Research
Volume 2011, Article ID 513905, 9 pages
Research Article

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

1Department of Biochemistry, Virginia Tech, Blacksburg, VA 26061, USA
2Enzyme Research and Drug Discovery Laboratory, Virginia Tech, Blacksburg, VA 26061, USA
3Fralin Life Science Institute, Virginia Tech, Blacksburg, VA 26061, USA

Received 2 April 2011; Accepted 20 May 2011

Academic Editor: Qi-Zhuang Ye

Copyright © 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.


Aspergillus 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.