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J. Biol. Chem., Vol. 280, Issue 12, 11295-11302, March 25, 2005

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The Functional Basis of Mycophenolic Acid Resistance in Candida albicans IMP Dehydrogenase^*

Gerwald A. Köhler¶, Xin Gong||, Stefan Bentink, Stephanie Theiss, Gina M. Pagani**, Nina Agabian, and Lizbeth Hedstrom||

From the ^||Department of Biochemistry and the ^**Graduate Program in Biophysics and Structural Biology, Brandeis University, Waltham, Massachusetts 02454-9110, the Research Center for Infectious Diseases, University of Würzburg, Röntgenring 11, D-97070 Würzburg, Germany, and the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143

Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to the same site as a mobile flap. In contrast, mizoribine monophosphate, the active metabolite of mizoribine, induces the closed conformation. Here, we report the characterization of IMPDH from wild-type and MPA-resistant strains of C. albicans. The wild-type enzyme displays significant differences from human IMPDHs, suggesting that selective inhibitors that could be novel antifungal agents may be developed. IMPDH from the MPA-resistant strain contains a single substitution (A251T) that is far from the MPA-binding site. The A251T variant was 4-fold less sensitive to MPA as expected. This substitution did not affect the k_cat value, but did decrease the K_m values for both substrates, so the mutant enzyme is more catalytically efficient as measured by the value of k_cat/K_m. These simple criteria suggest that the A251T variant would be the evolutionarily superior enzyme. However, the A251T substitution caused the enzyme to be 40-fold more sensitive to mizoribine monophosphate. This result suggests that A251T stabilizes the closed conformation, and this hypothesis is supported by further inhibitor analysis. Likewise, the MPA-resistant strain was more sensitive to mizoribine in cell culture. These observations illustrate the evolutionary challenge posed by the gauntlet of chemical warfare at the microbial level.

Received for publication, August 26, 2004 , and in revised form, December 30, 2004.

^* This work was supported in part by National Institutes of Health Grants GM54403 (to L. H.) and R01 AI33317 (to N. A.) and by Bundesministerium für Bildung und Forschung Grant O1 K18906-0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY864854.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S5.

^¶ Recipient of a Stipendienprogramm Infektionsforschung grant from the Bundesministerium für Bildung und Forschung.

To whom correspondence should be addressed: Dept. of Biochemistry, Brandeis University, MS 009, 415 South St., Waltham, MA 02454-9110. Tel.: 781-736-2333; Fax: 781-736-2349; E-mail: hedstrom{at}brandeis.edu.

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